Crispr system based antiviral therapy

ABSTRACT

The present invention offers a new approach for highly multiplexed, programmable antiviral therapies that directly target viral RNA, and can be flexibly adapted to target novel viruses or emerging outbreak pathogens. Class 2, type VI CRISPR system-based therapies can be used in combination with existing antiviral compounds for viruses where such compounds exist, either by increasing their efficacy or by preventing the evolution of specific drug resistance mutations. Perhaps most excitingly, if a virus evolves resistance to a specific guide RNA sequence, it is easy to switch to a different guide RNA sequence, or to design a new guide sequence to target the new mutation. Such approaches should prevent the widespread development of resistance to Class 2, type VI CRISPR system-based therapies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/530,029 filed Jul. 7, 2017. The entire contents of theabove-identified application are hereby fully incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersMH1007006 MH11049, and AI110818 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (“BROD_2810US_ST25.txt,”1,475,927 bytes, created on Jan. 24, 2020) is herein incorporated byreference in its entirety.

TECHNICAL FIELD

The subject matter disclosed herein is generally directed to the use ofCRISPR effector systems for use in treating, preventing, suppressing,and/or alleviating viral pathogenesis, infection, propagation, and/orreplication in a subject.

BACKGROUND

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity showextreme diversity of protein composition and genomic loci architecture.The CRISPR-Cas system loci has more than 50 gene families and there isno strictly universal genes indicating fast evolution and extremediversity of loci architecture. So far, adopting a multi-prongedapproach, there is comprehensive cas gene identification of about 395profiles for 93 Cas proteins. Classification includes signature geneprofiles plus signatures of locus architecture. A new classification ofCRISPR-Cas systems is proposed in which these systems are broadlydivided into two classes, Class 1 with multisubunit effector complexesand Class 2 with single-subunit effector modules exemplified by the Cas9protein. Novel effector proteins associated with Class 2 CRISPR-Cassystems may be developed as powerful genome engineering tools and theprediction of putative novel effector proteins and their engineering andoptimization is important.

The CRISPR-Cas adaptive immune system defends microbes against foreigngenetic elements via DNA or RNA-DNA interference. Recently, the Class 2type VI single-component CRISPR-Cas effector Cas13a, previously known asC2c2 (Shmakov et al. (2015) “Discovery and Functional Characterizationof Diverse Class 2 CRISPR-Cas Systems”; Molecular Cell 60:1-13; doi:http://dx.doi.org/10.1016/j.molcel.2015.10.008) was characterized as anRNA-guided Rnase (Abudayyeh et al. (2016), Science, [Epub ahead ofprint], June 2; “C2c2 is a single-component programmable RNA-guidedRNA-targeting CRISPR effector”; doi: 10.1126/science.aaf5573). It wasdemonstrated that C2c2 (e.g. from Leptotrichia shahii) provides robustinterference against RNA phage infection. Through in vitro biochemicalanalysis and in vivo assays, it was shown that C2c2 can be programmed tocleave ssRNA targets carrying protospacers flanked by a 3′ H (non-G)PAM. Cleavage is mediated by catalytic residues in the two conservedHEPN domains of C2c2, mutations in which generate a catalyticallyinactive RNA-binding protein. C2c2 is guided by a single crRNA and canbe re-programmed to deplete specific mRNAs in vivo. It was shown thatLshC2c2 can be targeted to a specific site of interest and can carry outnon-specific RNase activity once primed with the cognate target RNA.These results broaden our understanding of CRISPR-Cas systems anddemonstrate the possibility of harnessing Cas13, such as Cas13a, Cas13b,or Cas13c to develop a broad set of RNA-targeting tools.

While interference with phage infection in prokaryotes has beendemonstrated for LsCas13a, it is currently unknown if Cas13 mediatedantiviral therapy is feasible or even possible at all in a eukaryoticsetting. Indeed, the extreme differences between prokaryotes andeukaryotes, further confounded by the very nature of prokaryotic versuseukaryotic viruses, including etiology and pathogenesis, makesextrapolation from prokaryotic immunity to eukaryotic immunity highlyunpredictable.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY

Antiviral drugs do not exist for most emerging viruses, and availabledirect-acting antivirals, which include small molecules, shortinterfering RNAs, and antibodies, typically target a small number ofhighly mutable viral proteins or RNAs. This is problematic because RNAviruses evolve rapidly and can easily acquire resistance to existingtherapeutics. The present invention offers a new approach for highlymultiplexed, programmable antiviral therapies that directly target viralRNA, and can be flexibly adapted to target novel viruses or emergingoutbreak pathogens. Class 2, type VI CRISPR system-based therapies canbe used in combination with existing antiviral compounds for viruseswhere such compounds exist, either by increasing their efficacy or bypreventing the evolution of specific drug resistance mutations. Perhapsmost excitingly, if a virus evolves resistance to a specific guide RNAsequence, it is easy to switch to a different guide RNA sequence, ordesign a new guide sequence to target the new mutation. Such approachesshould prevent the widespread development of resistance to Class 2, typeVI CRISPR system-based therapies.

Current gold-standard pathogen diagnostics are often expensive, slow,and lack sufficient sensitivity to detect viral infections. Standardmolecular amplification methods, such as RT-qPCR, typically requirenucleic acid extraction and expensive thermocycling machinery.Immunoassays, such as ELISAs, can only detect single targets,cross-react to antigenically similar targets, and cannot be quicklydeveloped or updated to deal with new or evolving threats. By means ofexample, and without limitation, CRISPR-based detection/diagnosticplatforms, such as described in Grootenberg et al. (2017), “Nucleic aciddetection with CRISPR-Cas13/C2c2”, Science, 356(6336):438-442 cantransform the diagnosis of viral diseases with single-molecule detectionsensitivity and single nucleotide polymorphism specificity.

The present inventors have surprisingly found that Class 2, type VICRISPR systems are useful as an antiviral therapeutic or prophylactic.

The present invention has, among others, the following objectives: (1)Dissecting viral targets and viral evolution in response to anti-viraltherapy, including Class 2, type VI CRISPR system therapy, to enablerobust targeting. Methods and guides are identified which effectivelyinhibit viral pathogenesis, such as but not limited to viralreplication. In certain embodiments, the guides are selected whichreduce or avoid the evolution of viral resistance; (2) Multiplexed Class2, type VI CRISPR system-based viral therapeutics to evade viralevolution; (3) Detecting viral mutations using, for example, Class 2,type VI CRISPR system-based diagnostics. This includes diagnostics thatcan detect novel mutations that arise in the course of therapy as wellas mutations known to arise which lead to treatment resistance. Suchdiagnostics can be companion diagnostics to the therapeutic methods ofthe invention as described herein and can be used to select an initialanti-viral therapy and also to modify an anti-viral therapy in responseto resistant mutants that may emerge.

In certain aspects, a Class 2, type VI CRISPR system-based diagnostic isused for disease surveillance before, during, or after the course ofanti-viral therapy.

The present inventors have found that Class 2, type VI CRISPR systems,such as Cas13a, Cas13b, or Cas 13c have transformative potential as atool for rapidly formulating antiviral therapeutics. Furthermore, Class2, type VI CRISPR system-based therapies can be easily retargeted bychanging the guide RNA sequences, which can help combatting theevolution of therapy resistance. Resistance mutations can also berapidly, sensitively, and specifically detected, such as for instance,but not exclusively using the Cas13-based SHERLOCK platform (Grootenberget al. (2017), “Nucleic acid detection with CRISPR-Cas13/C2c2”, Science,356(6336):438-442). This is a major improvement relative to existingapproaches, which can take years to develop a single therapy for asingle virus and are difficult to reformulate. Thus, the approachesaccording to the present invention enable the development of many newantiviral therapeutics. Moreover, this is an opportunity to learn basicinformation about how viruses evolve to evade treatment.

In certain aspects, the present invention relates to development ofprogrammable, multiplexed viral therapeutics and sensitive viraldiagnostics. In certain aspects, the invention relates to determiningthe rules for Class 2, type VI CRISPR effector targeting and analyzingviral evolution in response to Class 2, type VI CRISPR system targeting.In certain aspects, the invention relates to development of multiplexedClass 2, type VI CRISPR system-based therapies, such as to thwart viralevolution. In certain aspects, the invention relates to Class 2, type VICRISPR system-based diagnostics, such as to detect viruses that couldcause outbreaks and viral mutations that may occur during outbreaks.

Accordingly, in one aspect, the invention provides methods for treating,preventing, suppressing, and/or alleviating viral pathogenesis,infection, propagation, and/or replication in a subject, comprisingadministering to a subject in need thereof a Class 2, type VI CRISPRsystem comprising (a) a Class 2, type VI CRISPR effector protein and/ora polynucleic acid encoding said effector protein and (b) one or moreguide RNAs and/or one or more polynucleic acids encoding said one ormore guide RNAs designed to bind to one or more target molecules of avirus. In certain embodiments, viremia or viral load or titer is reducedor suppressed.

In another aspect, the invention relates to a Class 2, type VI CRISPRsystem comprising (a) a Class 2, type VI CRISPR effector protein and/ora polynucleic acid encoding said effector protein and (b) one or moreguide RNAs and/or one or more polynucleic acids encoding said one ormore guide RNAs designed to bind to one or more target molecules of avirus for use in treating, preventing, suppressing, and/or alleviatingviral pathogenesis, infection, propagation and/or replication in asubject. In certain embodiments, viremia or viral load or titer isreduced or suppressed.

In a further aspect, the invention relates to a the use of Class 2, typeVI CRISPR system comprising (a) a Class 2, type VI CRISPR effectorprotein and/or a polynucleic acid encoding said effector protein and (b)one or more guide RNAs and/or one or more polynucleic acids encodingsaid one or more guide RNAs designed to bind to one or more targetmolecules of a virus for the manufacture of a medicament for treating,preventing, suppressing, and/or alleviating viral pathogenesis,infection, propagation and/or replication in a subject. In certainembodiments, viremia or viral load or titer is reduced or suppressed.

To provide better surveillance of key viral mutations in nature, thepresent invention provides Class 2, type VI CRISPR system-baseddiagnostics for detecting viral mutations, variations, or polymorphisms,such as natural variations, or such as that may occur or have occurredduring outbreaks. By means of example, and without limitation, a singlenucleotide polymorphism in LCMV that is associated with the phenotypicswitch between acute and persistent infection may be detected; thesingle nucleotide polymorphisms observed during an Ebola virus epidemicor a Zika virus epidemic may be detected, as well as the singlenucleotide polymorphisms that are responsible for drug resistance in HIVmay be detected. These tools enable to track viral evolution duringfuture outbreaks, and better understand the role of adaptive mutations.

In a further aspect, the invention relates to methods for detecting avirus, in particular in vitro methods for detecting a virus. In afurther aspect, the invention relates to methods for diagnosing a viralinfection, in particular in vitro methods for diagnosing a viralinfection. In a further aspect, the invention relates to methods formonitoring a viral infection, in particular in vitro methods formonitoring a viral infection. In a further aspect, the invention relatesto methods for detecting or monitoring viral pathogenesis, infection,propagation and/or replication, in particular in vitro methods fordetecting or monitoring viral pathogenesis, infection, propagationand/or replication. In a further aspect, the invention relates tomethods for detecting or monitoring viral evolution, in particular invitro methods for detecting or monitoring viral evolution. In a furtheraspect, the invention relates to methods for detecting or monitoringviral mutations or polymorphisms, in particular in vitro methods fordetecting or monitoring viral mutations or polymorphisms. In a furtheraspect, the invention relates to methods for detecting or monitoringdevelopment or evolution of viral resistance such as resistance againstthe therapeutics of the invention, in particular in vitro methods fordetecting or monitoring development or evolution of viral resistance,such as resistance to the therapeutics of the invention. In certainembodiments, these methods may involve CRISPR/Cas system based detectionsystems, such as described for instance in Gootenberg et al. (2017),“Nucleic acid detection with CRISPR-Cas13/C2c2”, Science,356(6336):438-442, which is incorporated herein by reference in itsentirety. The methods are however not limited to such CRISPR/Cas systembased detection systems. In certain embodiments, these methods arecomplementary diagnostic methods of the therapeutic methods of theinvention. In certain embodiments, these methods are companiondiagnostic methods of the therapeutic methods of the invention.

Given the high mutation rate of viruses, and in particular RNA viruses,monotherapy via individual guide RNAs may not completely inhibit viralpathogenesis, such as viral replication for extended periods of time. Todetermine the evolutionary response to Class 2, type VI CRISPRsystem-based therapy, in certain aspects, the invention relates tomethods to determine if particular regions of the viral genome are moreprone to evolving resistance to guide targeting regions of the viralgenome. Based on such analysis, suitable gRNAs can be selected.Advantageously, to further minimize development of viral resistance, theinvention in certain aspects relates to multiplexed approaches, in whichmultiple gRNAs are used to target particular viruses, particularstrains, or particular viral variants. For instance, by using severalguides, such as 2 or more, or 3 or more distinct guides, the evolutionof resistance to Class 2, type VI CRISPR system-based therapies can beminimized. In the rare case that any resistance were to be observed, theguide RNA sequences being used could easily be switched. For instance, aguide specifically targeting the emerging resistance mutations can bedesigned or a guide targeting an entirely different region of the viralgenome. Alternatively, if multiple guides are not sufficient to inhibitviral replication on their own, they can be used in combination withexisting therapeutics.

The invention further relates to polynucleic acids, vectors, vectorsystems, compositions, such as pharmaceutical compositions, comprisingClass 2, type VI CRISPR system comprising (a) a Class 2, type VI CRISPReffector protein and/or a polynucleic acid encoding said effectorprotein and (b) one or more guide RNAs and/or one or more polynucleicacids encoding said one or more guide RNAs designed to bind to one ormore target molecules of a virus, and their use in or for treating,preventing, suppressing, and/or alleviating viral pathogenesis,infection, propagation and/or replication in a subject. The inventionalso relates to methods for treating, preventing, suppressing, and/oralleviating viral pathogenesis, infection, propagation and/orreplication in a subject comprising administering such polynucleicacids, vectors, vector systems or compositions, as well as the use ofsuch polynucleic acids, vectors, vector systems or compositions for themanufacture of a medicament for treating, preventing, suppressing,and/or alleviating viral pathogenesis, infection, propagation and/orreplication in a subject. In certain embodiments, viremia or viral loador titer is reduced. An aspect of the invention is that the aboveelements are comprised in a single composition or comprised inindividual compositions.

In certain embodiments, the effector protein and/or guide RNA arecomprised in one or more polynucleic acid, such as a polynucleic acidencoding the effector protein and/or guide RNA. In certain embodiments,said polynucleic acid encoding said effector protein comprises aregulatory element operably linked to a polynucleic acid encoding saideffector protein. In certain embodiments, said polynucleic acid encodingsaid one or more guide RNAs comprises a regulatory element operablylinked to a polynucleic acid encoding said one or more guide RNAs. Incertain ebodiments, said polynucleic acid encoding said one or moreguide RNAs and/or said effector protein are comprised in one or morevectors, preferably (eukaryotic) expression vectors. In certainebodiments, said vector is a viral vector. In certain ebodiments, saidviral vector is an adenoviral vector, an AAV vector, or a retroviralvector.

In certain embodiments, the effector protein and/or guide RNA arecomprised in a polynucleic acid, preferably operably linked to aregulatory element, e.g. a promoter, such as a vector, wherein saideffector protein and/or guide RNA are being expressed or are capable ofbeing expressed constitutively or inducibly. In certain embodiments, theeffector protein and/or guide RNA are comprised in a polynucleic acid,preferably operably linked to a regulatory element, e.g. a promoter,such as a vector, wherein said effector protein and/or guide RNA arebeing expressed or are capable of being expressed in a tissue specificmanner.

In certain embodiments, the target molecule is, comprises, consists of,or consists essentially of a polynucleic acid. In certain embodiments,the target molecule is, comprises, consists of, or consists essentiallyof RNA. In certain embodiments, the target molecule is, comprises,consists of, or consists essentially of a viral target molecule. Incertain embodiments, the target molecule is, comprises, consists of, orconsists essentially of RNA. In certain embodiments, the target moleculeis, comprises, consists of, or consists essentially of a viral RNA. Incertain embodiments, the target molecule is, comprises, consists of, orconsists essentially of RNA. In certain embodiments, the target moleculeis, comprises, consists of, or consists essentially of a viral RNAtranscribed from a viral DNA.

The subject may be a human or animal subject. In particular embodiments,the subject is a mammalian subject. The virus may thus be a human oranimal virus. Alternatively, the virus may be a mammalian virus. Thevirus may be causative of human or animal disease. The virus may becausative of mammalian disease.

In certain embodiments, the virus is a plant virus.

In particular embodiments, the virus is an RNA virus. In furtherembdiments, the virus is a single stranded or double stranded RNA virus.In further embodiments, the virus is a positive sense RNA virus or anegative sense RNA virus or an ambisense RNA virus.

In certain embodiments, the one or more guide RNA binds to the codingstrand of the RNA. In certain embodiments, the guide RNA binds to thenon-coding strand of the RNA. In certain embodiments, the guide RNAbinds to viral genomic RNA (positive or negative sense or coding ornon-coding strand). In certain embodiments, the guide RNA binds totranscribed RNA (positive or negative sense or coding or non-codingstrand) from viral genomic DNA or transcribed RNA from a provirus.

In further embodiments, the virus is a Retroviridae virus, Lentiviridaevirus, Coronaviridae virus, a Picornaviridae virus, a Caliciviridaevirus, a Flaviviridae virus, a Togaviridae virus, a Bomaviridae, aFiloviridae, a Paramyxoviridae, a Pneumoviridae, a Rhabdoviridae, anArenaviridae, a Bunyaviridae, an Orthomyxoviridae, or a Deltavirus.

In particular embodiments, the virus is selected from the groupconsisting of Lymphocytic choriomeningitis virus, Coronavirus, HIV,SARS, Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, Yellow fevervirus, West Nile virus, Hepatitis C virus, Dengue fever virus, Zikavirus, Rubella virus, Ross River virus, Sindbis virus, Chikungunyavirus, Boma disease virus, Ebola virus, Marburg virus, Measles virus,Mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, Humanrespiratory syncytial virus, Rabies virus, Lassa virus, Hantavirus,Crimean-Congo hemorrhagic fever virus, Influenza and Hepatitis D virus.

In particular embodiments, the virus is a DNA virus. In furtherembdiments, the virus is a single stranded or double stranded DNA virus.In further embodiments, the virus is a positive sense DNA virus or anegative sense DNA virus or an ambisense DNA virus. In furtherembodiments, the virus is a Myoviridae, Podoviridae, Siphoviridae,Alloherpesviridae, Herpesviridae (including human herpes virus, andVaricella Zozter virus), Malocoherpesviridae, Lipothrixviridae,Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae(including African swine fever virus), Baculoviridae, Cicaudaviridae,Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae,Guttaviridae, Hytrosaviridae, Iridoviridae, Maseilleviridae,Mimiviridae, Nudiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae,Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae(including Simian virus 40, JC virus, BK virus), Poxviridae (includingCowpox and smallpox), Sphaerolipoviridae, Tectiviridae, Turriviridae,Dinodnavirus, Salterprovirus, or Rhizidovirus.

In certain embodiments, the effector protein comprises one or more HEPNdomains, preferablt two HEPN domains. In certain embodiments, the one ormore HEPN domains comprises a RxxxxH motif sequence. In certainembodiments, the RxxxH motif comprises a R{N/H/K]X1X2X3H sequence,preferably wherein X1 is R, S, D, E, Q, N, G, or Y, and X2 isindependently I, S, T, V, or L, and X3 is independently L, F, N, Y, V,I, S, D, E, or A.

In one example embodiment, the CRISPR system effector protein is aRNA-targeting effector protein. Example RNA-targeting effector proteinsinclude Cas13b, Cas13c, and C2c2 (now known as Cas13a). It will beunderstood that the term “C2c2” herein is used interchangeably with“Cas13a). In another example embodiment, the RNA-targeting effectorprotein is Cas13a, Cas13b, or Cas 13c. In other embodiments, the C2c2effector protein is from an organism of a genus selected from the groupconsisting of: Leptotrichia, Listeria, Corynebacter, Sutterella,Legionella, Treponema, Filifactor, Eubacterium, Streptococcus,Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium,Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia,Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, Campylobacter,and Lachnospira, or the C2c2 effector protein is an organism selectedfrom the group consisting of: Leptotrichia shahii, Leptotrichia. wadei,Listeria seeligeri, Clostridium aminophilum, Carnobacterium gallinarum,Paludibacter propionicigenes, Listeria weihenstephanensis, or the C2c2effector protein is a L. wadei F0279 or L. wadei F0279 (Lw2) C2C2effector protein.

In certain embodiments, Cas13a is selected from Cas13a from an organismselected from the Cas13a effector protein is from an organism selectedfrom the group consisting of: Leptotrichia shahii; Leptotrichia wadei(Lw2); Listeria seeligeri; Lachnospiraceae bacterium MA2020;Lachnospiraceae bacterium NK4A179; Clostridium aminophilum DSM 10710;Carnobacterium gallinarum DSM 4847; Carnobacterium gallinarum DSM 4847(second CRISPR Loci); Paludibacter propionicigenes WB4; Listeriaweihenstephanensis FSL R9-0317; Listeriaceae bacterium FSL M6-0635;Leptotrichia wadei F0279; Rhodobacter capsulatus SB 1003; Rhodobactercapsulatus R121; Rhodobacter capsulatus DE442; Leptotrichia buccalisC-1013-b; Herbinix hemicellulosilytica; Eubacterium rectale;Eubacteriaceae bacterium CHKCI004; Blautia sp. Marseille-P2398; andLeptotrichia sp. oral taxon 879 str. F0557, Lachnospiraceae bacteriumNK4A144; Chloroflexus aggregans; Demequina aurantiaca; Thalassospira sp.TSL5-1; Pseudobutyrivibrio sp. OR37; Butyrivibrio sp. YAB3001; Blautiasp. Marseille-P2398; Leptotrichia sp. Marseille-P3007; Bacteroidesihuae; Porphyromonadaceae bacterium KH3CP3RA; Listeria riparia; andInsolitispirillum peregrinum.

In certain embodiments, the effector protein cleaves the targetmolecule. In certain embodiments, the effector molecule cleaves thetarget RNA. In certain embodiments, the effector protein comprises oneor more mutations. In certain embodiments, the one or more mutationsaffect effector protein catalytic activity, stability, and/orspecificity.

In certain ebodiments, the effector protein is or comprises a fusionprotein. In certain ebodiments, the effector protein is a fusion proteinwith a heterologous domain. In certain embdiments, the effector proteincomprises a nuclear localization signal (NLS) or a nuclear export signal(NES). In certain embodiments the effector protein comprises aheterologous nuclear localization signal (NLS) or a nuclear exportsignal (NES).

In certain embodiments, the effector protein is codon optimized. It willbe understood that codon optimization may be species dependent.

In certain embodiments, the guide RNA comprises one or more, preferablyone, mismatch with the target sequence. In certain embodiments, theguide RNA comprises one or more, preferably one, synthetic mismatch withthe target sequence. In certain embodiments, said mismatch is up- ordownstream of a SNP or other single nucleotide variation in said targetmolecule. In certain embodiments, the guide RNAs comprise a pan-viralguide RNA set that targets each virus and/or viral strain in a set ofviruses.

In certain embodiments, more than one guide RNA is provided. In certainembodiments, the guide RNAs comprise a pan-viral guide RNA set thattargets each virus and/or viral strain in a set of viruses. Forinstance, a panel of guide RNAs may be provided which collectivelyrecognize different strains of a particular virus.

These and other aspects, objects, features, and advantages of theexample embodiments will become apparent to those having ordinary skillin the art upon consideration of the following detailed description ofillustrated example embodiments.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. All rights to explicitly disclaim anyembodiments that are the subject of any granted patent(s) of applicantin the lineage of this application or in any other lineage or in anyprior filed application of any third party is explicitly reserved.Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B—Show location of designed guide RNAs along the segmentedLCMV genome. (FIG. 1A) Guide RNAs S1-S6 are complementary to the LCMV SRNA (of which S1 and S6 bind respectively to the 5′ UTR and 3′UTR).(FIG. 1B) Guide RNAs L1-L6 are complementary to the LCMV L RNA (of whichL1 and L6 bind respectively to the 5′ UTR and 3′UTR). All guide RNAsbind to the coding strand.

FIG. 2—Cas13a and LCMV-specific guide RNA expression decreases LCMVreplication in cell culture. (FIG. 2A) HEK293FT cells were transfectedwith plasmids expressing Cas13a and single guide RNAs targeting LCMV ornon-targeting controls. 24 hours post-transfection, cells were infectedwith LCMV at an MOI of 5, and viral titers were measured 48 hourspost-infection using RT-qPCR of viral RNA in the culture supernatant.(FIG. 2B) Inhibition of viral replication. Empty vector, off-target #1and off-target #2 are considered negative controls. S1-S5 and L1-L6target various regions of the LCMV genome. Error bars indicate 1standard deviation based on n=3-6 biological replicates.

FIG. 3—Inhibition of viral replication. LCMV infected mammalian 293FTcells were transfected with the indicated C2c2 and guide plasmids. Plotsrepresent RT-qPCR values as genome equivalents per microliter with a barfor each transfected guide plasmid. Empty vector, off-target #1 andoff-target #2 are considered negative controls. S1-S5 and L1-L6 targetvarious regions of the LCMV genome. Error bars indicate 1 standarddeviation based on n=6 biological replicates.

FIG. 4—Combinations of multiple guides enhance the Cas13a-mediatedinhibition of LCMV replication. HEK293FT cells were transfected withplasmids expressing Cas13a and one or more guide RNAs targeting LCMV ornon-targeting controls. LCMV infection was performed 24 hourspost-transfection at an MOI of 5, and viral titers were measured 48hours post infection usting RT-qPCR of viral RNA in the culturesupernatant. Empty vector, off-target #1 and off-target #2 areconsidered negative controls. Error bars indicate one standard deviationbased on n=6 biological replicates.

FIG. 5—Fraction of guides reducing viral replication. Mean GFPfluorescence 48 hours post LCMV infection was calculated from 3replicates for all quides. Fold-change for each LCMV targeting guide wascalculated as the ratio of the mean fluorescence of the control guideover the LCMV targeting guide. P values were calculated using a 2tailed, unpaired t.test. Targeting guides were considered any guide witha p value less than or equal to 0.05 and fold change (FC) greater thanor equal to 2. The pie chart plots the data displayed in the table withwedges corresponding to the non-coding and coding region of LCMV's 4proteins. Remaining guides are those LCMV targeting guides that do notpass the p value and FC threshold.

FIG. 6—Distribution of targeting efficiency of targeting guides. Thedistribution of fold change of GFP fluorescence (control guide over LCMVtargeting guide) for guides that passed a p-value threshold of 0.05. Notshown on this graph, 8 guides with GFP fluorescence reduction greaterthan 50 fold (* 8 guides show reduction >50 fold).

FIGS. 7A and 7B—Representative images (3 replicates for each guide)illustrating the reduction of GFP (i.e. LCMV replication) for (FIG. 7B)Guide targeting the coding region of L (#104) compared to (FIG. 7A) thecontrol (empty guide vector). Images were taken 48 hours post LCMVinfection at magnification of 4×. Fold change 2.72, p value 0.047.

FIG. 8—Fold change of GFP fluorescence 48 hours post infection ofcontrol guide over LCMV-targeting guide for all guides that passed ap-value threshold of 0.05 and fold change threshold of 2. Each positionon the x-axis is a guide that was tested in LCMV full-genome screen. Anyguide that did not pass this threshold was plotted as 1. For any guidewith fluorescence less than or at background, the fold change is set asthe maximum fold change observed.

FIGS. 9A-9C—Cas13a-based diagnostics can sensitively detect Zika virusnucleic acid. Zika virus cDNA was serially diluted in healthy humanurine and water, inactivated endogenous human RNases, and used theSHERLOCK protocol to quantify viral cDNA (FIG. 9A). Several cDNA sampleswere also tested from patient urine or serum (FIG. 9B), and acombination of guide RNAs were used to distinguish between patientsample collected from different countries during the Zika virus outbreak(FIG. 9C). Error bars indicate one standard deviation. Abbreviations:DOM=Dominican Republic, DOMUSA=Dominican Republic/USA, HND=Honduras,USA=United State of America.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Definitions of common termsand techniques in molecular biology may be found in Molecular Cloning: ALaboratory Manual, 2^(nd) edition (1989) (Sambrook, Fritsch, andManiatis); Molecular Cloning: A Laboratory Manual, 4^(th) edition (2012)(Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (AcademicPress, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B.D. Hames, and G. R. Taylor eds.): Antibodies, A Laboraotry Manual (1988)(Harlow and Lane, eds.): Antibodies A Laboraotry Manual, 2^(nd) edition2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney,ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008(ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829);Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology andMolecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed.,John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Janvan Deursen, Transgenic Mouse Methods and Protocols, 2^(nd) edition(2011).

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The term “optional” or “optionally” means that the subsequent describedevent, circumstance or substituent may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The terms “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, are meant to encompass variations of and from thespecified value, such as variations of +/−10% or less, +/−5% or less,+/−1% or less, and +/−0.1% or less of and from the specified value,insofar such variations are appropriate to perform in the disclosedinvention. It is to be understood that the value to which the modifier“about” or “approximately” refers is itself also specifically, andpreferably, disclosed.

Reference throughout this specification to “one embodiment”, “anembodiment,” “an example embodiment,” means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,appearances of the phrases “in one embodiment,” “in an embodiment,” or“an example embodiment” in various places throughout this specificationare not necessarily all referring to the same embodiment, but may.Furthermore, the particular features, structures or characteristics maybe combined in any suitable manner, as would be apparent to a personskilled in the art from this disclosure, in one or more embodiments.Furthermore, while some embodiments described herein include some butnot other features included in other embodiments, combinations offeatures of different embodiments are meant to be within the scope ofthe invention. For example, in the appended claims, any of the claimedembodiments can be used in any combination.

Specific reference is made to U.S. Provisional Application No.62/471,931 filed Mar. 15, 2017 and entitled “CRISPR Effector SystemBased Diagnostics” and U.S. Provisional Application No. 62/484,857 filedon Apr. 12, 2017 and entitled “CRISPR Effector System Based Diagnosticsfor Virus Detection.”

All publications, published patent documents, and patent applicationscited herein are hereby incorporated by reference to the same extent asthough each individual publication, published patent document, or patentapplication was specifically and individually indicated as beingincorporated by reference

Microbial Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systemscontain programmable endonucleases, such as Cas9 and Cpf1 (Shmakov etal., 2017; Zetsche et al., 2015). Although both Cas9 and Cpf1 targetDNA, single effector RNA-guided RNases have been recently discovered(Shmakov et al., 2015) and characterized (Abudayyeh et al., 2016;Smargon et al., 2017), including C2c2, providing a platform for specificRNA sensing. RNA-guided RNases can be easily and convenientlyreprogrammed using CRISPR RNA (crRNAs) to cleave target RNAs.

In an aspect, the present invention relates to the use of the CRISPRsystem, in particular a Class 2, type VI CRISPR system, as an antiviraltherapy or prophylactic (e.g. immunization), and/or as a viraldiagnostic. The embodiments disclosed herein utilize RNA targetingeffectors to treat and prevent infection by a viral pathogen in asubject. It has been found that RNA-targeting CRISPR proteins can beused to suppress different stages of infection of a eukaryotic cell by avirus. This implies that these CRISPR systems can be used to treat orprevent diseases caused by such viruses. This is of interest not only inhuman antiviral therapy, for diseases such as Ebola hemorrhoragic fever,SARS, hepatitis C, West Nile fever, polio and measles, but also for farmanimals such as sheep and cows suffering from diseases such as Bovineviral diarrhea and Parainfluenza-3 virus-caused respiratory infections.In certain embodiments, the CRISPR systems according to the invention asdescribed herein are used or can be used for immunization.

In one aspect, the embodiments disclosed herein are directed to methodsfor treating, preventing, suppressing, and/or alleviating infection,propagation, replication of and/or pathogenesis caused by a virus in asubject, comprising administering to a subject in need thereof a Class2, type VI CRISPR system comprising an effector protein or a polynucleicacid encoding an effector protein and one or more guide RNAs or one ormore polynucleic acids encoding one or more guide RNAs designed to bindto one or more target molecules of said virus. The polynucleic acidencoding said one or more guide RNAs and/or the effector protein may becomprised in one or more vector, which may be the same or differentvectors, preferably (eukaryotic) expression vectors. Accordingly, theapplication provides a Class 2, type VI CRISPR system comprising aneffector protein or a polynucleic acid encoding an effector protein andone or more guide RNAs or one or more polynucleic acids encoding one ormore guide RNAs designed to bind to one or more target molecules of avirus for use in treating, preventing, suppressing, and/or alleviatinginfection, propagation, replication of and/or pathogenesis caused by avirus in a subject. Also, the invention provides pharmaceuticalcompositions comprising the CRISPR system as defined herein, for use intreating, preventing, suppressing, and/or alleviating infection,propagation, replication of and/or pathogenesis caused by a virus in asubject.

In an aspect, the invention provides methods and compositions formodulating, e.g., reducing, (protein) expression of a (viral) target RNAin cells. In the subject methods, a CRISPR system of the invention isprovided that interferes with transcription, stability, and/ortranslation of an RNA.

In certain embodiments, an effective amount of CRISPR system is used tocleave RNA or otherwise inhibit RNA expression. In this regard, thesystem has uses similar to siRNA and shRNA, thus can also be substitutedfor such methods. The method includes, without limitation, use of aCRISPR system as a substitute for e.g., an interfering ribonucleic acid(such as an siRNA or shRNA) or a transcription template thereof, e.g., aDNA encoding an shRNA. The CRISPR system is introduced into a targetcell, e.g., by being administered to a mammal that includes the targetcell,

Advantageously, a CRISPR system of the invention is specific. Forexample, whereas interfering ribonucleic acid (such as an siRNA orshRNA) polynucleotide systems are plagued by design and stability issuesand off-target binding, a CRISPR system of the invention can be designedwith high specificity.

In certain embodiments, the systems, compositions, polynucleic acids,vector and vector systems, and methods, disclosed herein are useful fordiagnosing and/or treating viral pathogenesis, infection, propagation,and/or replication in a subject. In certain embodiments, the systems,compositions, polynucleic acids, vector and vector systems, and methods,disclosed herein are useful for preventing viral pathogenesis,infection, propagation, and/or replication in a subject. In certainembodiments, the systems, compositions, polynucleic acids, vector andvector systems, and methods, disclosed herein are useful for suppressingviral pathogenesis, infection, propagation, and/or replication in asubject. In certain embodiments, the systems, compositions, polynucleicacids, vector and vector systems, and methods, disclosed herein areuseful for alleviating viral pathogenesis, infection, propagation,and/or replication in a subject. In certain example embodiments, thesystems, compositions, polynucleic acids, vector and vector systems, andmethods, disclosed herein are useful for immunization against a virus.In certain example embodiments, the systems, compositions, polynucleicacids, vector and vector systems, and methods, disclosed herein areuseful for suppressing or alleviating viremia or reducing viral load ortiter in a subject. In certain embodiments, the systems, compositions,polynucleic acids, vector and vector systems, and methods, disclosedherein are useful for treating viral pathogenesis in a subject. Incertain embodiments, the systems, compositions, polynucleic acids,vector and vector systems, and methods, disclosed herein are useful forpreventing viral pathogenesis in a subject. In certain embodiments, thesystems, compositions, polynucleic acids, vector and vector systems, andmethods, disclosed herein are useful for suppressing viral pathogenesisin a subject. In certain embodiments, the systems, compositions,polynucleic acids, vector and vector systems, and methods, disclosedherein are useful for alleviating viral pathogenesis in a subject. Incertain embodiments, the systems, compositions, polynucleic acids,vector and vector systems, and methods, disclosed herein are useful fortreating viral infection in a subject. In certain embodiments, thesystems, compositions, polynucleic acids, vector and vector systems, andmethods, disclosed herein are useful for preventing viral infection in asubject. In certain embodiments, the systems, compositions, polynucleicacids, vector and vector systems, and methods, disclosed herein areuseful for suppressing viral infection in a subject. In certainembodiments, the systems, compositions, polynucleic acids, vector andvector systems, and methods, disclosed herein are useful for alleviatingviral infection in a subject. In certain embodiments, the systems,compositions, polynucleic acids, vector and vector systems, and methods,disclosed herein are useful for treating viral propagation in a subject.In certain embodiments, the systems, compositions, polynucleic acids,vector and vector systems, and methods, disclosed herein are useful forpreventing viral propagation in a subject. In certain embodiments, thesystems, compositions, polynucleic acids, vector and vector systems, andmethods, disclosed herein are useful for suppressing viral propagationin a subject. In certain embodiments, the systems, compositions,polynucleic acids, vector and vector systems, and methods, disclosedherein are useful for alleviating or reducing viral propagation in asubject. In certain embodiments, the systems, compositions, polynucleicacids, vector and vector systems, and methods, disclosed herein areuseful for treating viral replication in a subject. In certainembodiments, the systems, compositions, polynucleic acids, vector andvector systems, and methods, disclosed herein are useful for preventingviral replication in a subject. In certain embodiments, the systems,compositions, polynucleic acids, vector and vector systems, and methods,disclosed herein are useful for suppressing viral replication in asubject. In certain embodiments, the systems, compositions, polynucleicacids, vector and vector systems, and methods, disclosed herein areuseful for alleviating or reducing viral replication in a subject.

In certain embodiments, the virus is a pathogenic virus. In certaimembodiments, the virus is an opportunistic pathogenic virus. In certainembodiments, the virus is causative of a disease, preferably in a humanor animal subject, such as a mammalian subject. In certain embodiments,the virus may cause acute disease (e.g. in human or animal or inmammal). In certain embodiments, the virus may cause chronic disease(e.g. in human or animal or in mammal). In certain embodiments, thevirus may be dormant or in a dormant state. In certain embodiments, thevirus may be latent or in latency or a latent state. In certainembodiments, the virus may be lysogenic or in a lysogenic state. Incertain embodiments, the virus may be lytic or in a lytic state. Incertain embodiments, the virus may be a provirus. In certain embodimentsthe virus may be episomal. In certaim embodiments, the virus may be alatent provirus. In certain embodiments, the virus may be a latentepisomal virus.

In certain embodiments, the systems, compositions, polynucleic acids,vector and vector systems, and methods, disclosed herein are useful forinducing or maintaining viral latency or dormancy. In certainembodiments, the systems, compositions, polynucleic acids, vector andvector systems, and methods, disclosed herein are useful for preventingor reducing viral activation and/or viral shedding.

In certain example embodiments, the systems, compositions, polynucleicacids, vector and vector systems, and methods, disclosed herein areuseful for treating, preventing, suppressing, and/or alleviating viralpathogenesis, infection, propagation, and/or replication in a subject,or for immunization, or for reducing viremia or viral load or titer in asubject. In certain example embodiments, the systems, compositions,polynucleic acids, vector and vector systems, and methods, disclosedherein are useful for diagnosing a virus. The virus may be a DNA virus(single or double stranded, positive or negative sense or ambisense) oran RNA virus (single or double stranded, positive or negative sense orambisense). In certain embodiments, the virus is Ebola, measles, SARS,Chikungunya, hepatitis, Marburg, yellow fever, MERS, Dengue, Lassa,influenza, rhabdovirus or HIV. A hepatitis virus may include hepatitisA, hepatitis B, or hepatitis C. An influenza virus may include, forexample, influenza A or influenza B. An HIV may include HIV 1 or HIV 2.In certain example embodiments, the virus may be a human respiratorysyncytial virus, Sudan ebola virus, Bundibugyo virus, Tai Forest ebolavirus, Reston ebola virus, Achimota, Aedes flavivirus, Aguacate virus,Akabane virus, Alethinophid reptarenavirus, Allpahuayo mammarenavirus,Amapari mmarenavirus, Andes virus, Apoi virus, Aravan virus, Aroa virus,Arumwot virus, Atlantic salmon paramyoxivirus, Australian batlyssavirus, Avian bornavirus, Avian metapneumovirus, Avianparamyoxviruses, penguin or Falkland Islandsvirus, BK polyomavirus,Bagaza virus, Banna virus, Bat hepevirus, Bat sapovirus, Bear Canonmammarenavirus, Beilong virus, Betacoronoavirus, Betapapillomavirus 1-6,Bhanja virus, Bokeloh bat lyssavirus, Borna disease virus, Bourbonvirus, Bovine hepacivirus, Bovine parainfluenza virus 3, Bovinerespiratory syncytial virus, Brazoran virus, Bunyamwere virus,Caliciviridae virus. California encephalitis virus, Candiru virus,Canine distemper virus, Canaine pneumovirus, Cedar virus, Cell fusingagent virus, Cetacean morbillivirus, Chandipura virus, Chaoyang virus,Chapare mammarenavirus, Chikungunya virus, Colobus monkeypapillomavirus, Colorado tick fever virus, Cowpox virus, Crimean-Congohemorrhagic fever virus, Culex flavivirus, Cupixi mammarenavirus, Denguevirus, Dobrava-Belgrade virus, Donggang virus, Dugbe virus, Duvenhagevirus, Eastern equine encephalitis virus, Entebbe bat virus, EnterovirusA-D, European bat lyssavirus 1-2, Eyach virus, Feline morbillivirus,Fer-de-Lance paramyxovirus, Fitzroy River virus, Flaviviridae virus,Flexal mammarenavirus, GB virus C, Gairo virus, Gemycircularvirus, Gooseparamyoxiviurs SF02, Great Island virus, Guanarito mammarenavirus,Hantaan virus, Hantavirus Z10, Heartland virus, Hendra virus, HepatitisA/B/C/E, Hepatitis delta virus, Human bocavirus, Human coronavirus,Human endogenous retrovirus K, Human enteric coronavirus, Humangential-associated circular DNA virus-1, Human herpesvirus 1-8, Humanimmunodeficiency virus 1/2, Huan mastadenovirus A-G, Humanpapillomavirus, Human parainfluenza virus 1-4, Human paraechovirus,Human picobirnavirus, Human smacovirus, Ikoma lyssavirus, Ilheus virus,Influenza A-C, Ippy mammarenavirus, Irkut virus, J-virus, JCpolyomavirus, Japanses encephalitis virus, Junin mammarenavirus, KIpolyomavirus, Kadipiro virus, Kamiti River virus, Kedougou virus,Khujand virus, Kokobera virus, Kyasanur forest disease virus, Lagos batvirus, Langat virus, Lassa mammarenavirus, Latino mammarenavirus,Leopards Hill virus, Liao ning virus, Ljungan virus, Lloviu virus,Louping ill virus, Lujo mammarenavirus, Luna mammarenavirus, Lunk virus,Lymphocytic choriomeningitis mammarenavirus, Lyssavirus Ozernoe,MSSI2\.225 virus, Machupo mammarenavirus, Mamastrovirus 1, Manzanillavirus, Mapuera virus, Marburg virus, Mayaro virus, Measles virus,Menangle virus, Mercadeo virus, Merkel cell polyomavirus, Middle Eastrespiratory syndrome coronavirus, Mobala mammarenavirus, Modoc virus,Moijang virus, Mokolo virus, Monkeypox virus, Montana myotisleukoenchalitis virus, Mopeia lassa virus reassortant 29, Mopeiamammarenavirus, Morogoro virus, Mossman virus, Mumps virus, Murinepneumonia virus, Murray Valley encephalitis virus, Nariva virus,Newcastle disease virus, Nipah virus, Norwalk virus, Norway rathepacivirus, Ntaya virus, O'nyong-nyong virus, Oliveros mammarenavirus,Omsk hemorrhagic fever virus, Oropouche virus, Parainfluenza virus 5,Parana mammarenavirus, Parramatta River virus,Peste-des-petits-ruminants virus, Pichande mammarenavirus,Picornaviridae virus, Pirital mammarenavirus, Piscihepevirus A, Procineparainfluenza virus 1, porcine rubulavirus, Powassan virus, PrimateT-lymphotropic virus 1-2, Primate erythroparvovirus 1, Punta Toro virus,Puumala virus, Quang Binh virus, Rabies virus, Razdan virus, Reptilebornavirus 1, Rhinovirus A-B, Rift Valley fever virus, Rinderpest virus,Rio Bravo virus, Rodent Torque Teno virus, Rodent hepacivirus, RossRiver virus, Rotavirus A-I, Royal Farm virus, Rubella virus, Sabiamammarenavirus, Salem virus, Sandfly fever Naples virus, Sandfly feverSicilian virus, Sapporo virus, Sathuperi virus, Seal anellovirus,Semliki Forest virus, Sendai virus, Seoul virus, Sepik virus, Severeacute respiratory syndrome-related coronavirus, Severe fever withthrombocytopenia syndrome virus, Shamonda virus, Shimoni bat virus,Shuni virus, Simbu virus, Simian torque teno virus, Simian virus 40-41,Sin Nombre virus, Sindbis virus, Small anellovirus, Sosuga virus,Spanish goat encephalitis virus, Spondweni virus, St. Louis encephalitisvirus, Sunshine virus, TTV-like mini virus, Tacaribe mammarenavirus,Taila virus, Tamana bat virus, Tamiami mammarenavirus, Tembusu virus,Thogoto virus, Thottapalayam virus, Tick-borne encephalitis virus,Tioman virus, Togaviridae virus, Torque teno canis virus, Torque tenodouroucouli virus, Torque teno felis virus, Torque teno midi virus,Torque teno sus virus, Torque teno tamarin virus, Torque teno virus,Torque teno zalophus virus, Tuhoko virus, Tula virus, Tupaiaparamyxovirus, Usutu virus, Uukuniemi virus, Vaccinia virus, Variolavirus, Venezuelan equine encephalitis virus, Vesicular stomatitisIndiana virus, WU Polyomavirus, Wesselsbron virus, West Caucasian batvirus, West Nile virus, Western equine encephalitis virus, WhitewaterArroyo mammarenavirus, Yellow fever virus, Yokose virus, Yug Bogdanovacvirus, Zaire ebolavirus, Zika virus, or Zygosaccharomyces bailii virus Zviral sequence. Examples of RNA viruses that may be detected include oneor more of (or any combination of) Coronaviridae virus, a Picornaviridaevirus, a Caliciviridae virus, a Flaviviridae virus, a Togaviridae virus,a Bornaviridae, a Filoviridae, a Paramyxoviridae, a Pneumoviridae, aRhabdoviridae, an Arenaviridae, a Bunyaviridae, an Orthomyxoviridae, ora Deltavirus. In certain example embodiments, the virus is Coronavirus,SARS, Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, Yellow fevervirus, West Nile virus, Hepatitis C virus, Dengue fever virus, Zikavirus, Rubella virus, Ross River virus, Sindbis virus, Chikungunyavirus, Borna disease virus, Ebola virus, Marburg virus, Measles virus,Mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, Humanrespiratory syncytial virus, Rabies virus, Lassa virus, Hantavirus,Crimean-Congo hemorrhagic fever virus, Influenza, or Hepatitis D virus.

In certain embodiments, the virus is a virus listed in Table 1 below, ora virus of the indicated genus/family.

TABLE 1 Virus Genus, Family Host Transmission Disease Adeno-associatedvirus Dependovirus Human, vertebrates Respiratory None Aichi virusKobuvirus, Picornaviridae Human Fecal-oral Gastroenteritis Australianbat lyssavirus Lyssavirus, Rhabdoviridae Human, bats Zoonosis, animalbite Fatal encephalitis BK polyomavirus Polyomavirus, PolyomaviridaeHuman Respiratory fluids or urine None Banna virus Scadomavirus,Reoviridae Human, cattle, pig, mosquitoes Zoonosis, arthropod biteEncephalitis Barmah forest virus Alphavirus, Togaviridae Human,marsupials, mosquitoes Zoonosis, arthropod bite Fever, joint painBunyamwera virus Orthobunyavirus, Bunvaviridae Human, mosquitoesZoonosis, arthropod bite Encephalitis Bunyavirus La CrosseOrthobunyavirus, Bunyaviridae Human, deer, mosquitoes, Zoonosis,arthropod bite Encephalitis tamias Bunyavirus snowshoe hareOrthobunyavirus, Bunyaviridae Human, rodents, mosquitoes Zoonosis,arthropod bite Encephalitis Cercopithecine herpesvirusLymphocryptovirus, Herpesviridae Human, monkeys Zoonosis, animal biteEncephalitis Chandipura virus Vesiculovirus, Rhabdoviridae Human,sandflies Zoonosis, athropod bite Encephalitis Chikungunya virusAlphavirus, Togaviridae Human, monkeys, mosquitoes Zoonosis, arthropodbite Fever, joint pain Cosavirus A Cosavirus, Picornaviridae HumanFecal-oral (probable) — Cowpox virus Orthopoxvirus, Poxviridae Human,mammals Zoonosis, contact None Coxsackievirus Entcrovirus,Picornaviridae Human Fecal-oral Meningitis, myocarditis, paralysisCrimean-Congo Nairovirus, Bunyaviridae Human, vertebrates, ticksZoonosis, arthropod bite Hemorrhagic fever hemorrhagic fever virusDengue virus Flavivirus, Flaviviridae Human, mosquitoes Zoonosis,arthropod bite Hemorrhagic fever Dhori virus Thogotovirus,Orthomyxoviridae Human, ticks Zoonosis, arthropod bite Fever,encephalitis Dugbe virus Nairovirus, Bunyaviridae Human, ticks Zoonosis,arthropod bite Thrombocytopaenia Duvenhage virus Lyssavirus,Rhabdoviridae Human, mammals Zoonosis, animal bite Fatal encephalitisEastern equine Alphavirus, Togaviridae Human, birds, mosquitoesZoonosis, arthropod bite Encephalitis encephalitis virus EbolavirusEbolavirus, Filoviridae Human, monkeys, bats Zoonosis, contactHemorrhagic fever Echovirus Enterovirus, Picornaviridae Human Fecal-oralCommon cold Encephalomyocar ditis virus Cardiovirus, PicornaviridaeHuman, mouse, rat, pig Zoonosis Encephalitis Epstein-Barr virusLymphocryptovirus, Herpesviridae Human Contact, saliva MononucleosisEuropean bat lyssavirus Lyssavirus, Rhabdovirus Human, bats Zoonosis,animal bite Fatal encephalitis GB virus C/Hepatitis G virus Pegivirus,Flaviviridae Human Blood, occasionally sexual None Hantaan virusHantavirus, Bunyaviridae Human, rodents Zoonosis, urine, saliva Renal orrespiratory svndrome Hendra virus Henipavirus, paramyxoviridae Human,horse, bats Zoonosis, animal bite Encephalitis Hepatitis A virusHepatovirus, picornaviridae Human Fecal-oral Hepatitis Hepatitis B virusOrthohepadnavirus, Hepadnaviridae Human, Chimpanzees Sexual contact,blood Hepatitis Hepatitis C virus Hepacivirus, Flaviviridae HumanSexual, blood Hepatitis Hepatitis E virus Hepevirus, Unassigned Human,pig, monkeys, Zoonosis, food Hepatitis some rodents, chicken Hepatitisdelta virus Deltavirus, Unassigned Human Sexual contact, blood HepatitisHorsepox virus Orthopoxvirus, Poxviridae Human, horses Zoonosis, contactNone Human adenovirus Mastadenovirus, Adenoviridae Human Respiratory,fecal-oral Respiratory Human astrovirus Mamastrovirus, AstroviridaeHuman Fecal-oral Gastroenteritis Human coronavirus Alphacoronavirus,Coronaviridae Human Respiratory Respiratory Human cytomegalovirusCytomegalovirus, Herpesviridae Human Contact, urine, salivaMononucleosis, pneumonia Human enterovirus 68, 70 Enterovirus,Picornaviridae Human Fecal-oral Diarrhea, neurological disorder Humanherpesvirus 1 Simplexvirus, Herpesviridae Human Sexual contact, salivaSkin lesions Human herpesvirus 2 Simplexvirus, Herpesviridae HumanSexual contact, saliva Skin lesions Human herpesvirus 6 Roseolovirus,Herpesviridae Human Respiratory, contact Skin lesions Human herpesvirus7 Roseolovirus, Herpesviridae Human Respiratory, contact Skin lesionsHuman herpesvirus 8 Rhadinovirus, Herpesviridae Human Sexual contact,saliva Skin lymphoma Human immunodeficiency virus Lentivirus,Retroviridae Human Sexual contact, blood AIDS Human papillomavirus 1Mupapillomavirus, Human Contact Skin warts Papillomaviridae Humanpapillomavirus 2 Alphapapillomavirus, Human Contact Skin wartsPapillomaviridae Human papillomavirus 16,18 Alphapapillomavirus, HumanSexual Genital warts, Papillomaviridae cervical cancer Humanparainfluenza Respirovirus, Paramyxoviridae Human RespiratoryRespiratory Human parvovirus B19 Erythrovirus, Parvoviridae HumanRespiratory Skin lesion Human respiratory Pneumovirus, ParamyxoviridaeHuman Respiratory Respiratory syncytial virus Human rhinovirusEnterovirus Human Respiratory Respiratory Human SARS coronavirusBetacoronavirus, Coronaviridae Human, palm civet Zoonosis RespiratoryHuman spumaretrovirus Spumavirus, Retroviridae Human Contact, salivaNone Human T-lymphotropic virus Deltaretrovirus, Retroviridae HumanSexual contact, Leukemia maternal-neonatal Human torovirus Torovirus,Coronaviridae Human Fecal-oral Gastroenteritis Influenza A virusInfluenzavirus A, Human, birds, pigs Respiratory or Zoonosis, FluOrthomyxoviridae animal contact Influenza B virus Influenzavirus B,Human Respiratory Flu Orthomyxoviridae Influenza C virus InfluenzavirusC, Human Respiratory Flu Orthomyxoviridae Isfahan virus Vesiculovirus,Rhabdoviridae Human, sandflies, gerbils Zoonosis, arthropod biteUndocumented, encephalitis JC polyomavirus Polyomavirus, PolvomaviridaeHuman Fecal-oral or urine Encephalitis Japanese encephalitis virusFlavivirus, Flaviviridae Human, horses, birds, Zoonosis, arthropod borneEncephalitis mosquitoes Junin arenavirus Arenavirus, Arenaviridae Human,rodents Zoonosis, fomite Hemorrhagic fever KI Polyomavirus Polyomavirus,Polyomaviridae Human Fecal-oral or urine Encephalitis Kunjin virusFlavivirus, Flaviviridae Human, horses, birds, Zoonosis, arthropod borneEncephalitis mosquitoes Lagos bat virus Lyssavirus, Rhabdoviridae Human,mammals Zoonosis, animal bite Fatal encephalitis Lake Victoriamarburgvirus Marburgvirus, Filoviridae Human, monkeys, bats Zoonosis,fomite Hemorrhagic fever Langat virus Flavivirus, Flaviviridae Human,ticks Zoonosis, arthropod borne Encephalitis Lassa virus Arenavirus,Arenaviridae Human, rats Zoonosis, fomites Hemorrhagic fever Lordsdalevirus Norovirus, Caliciviridae Human Fecal-oral Gastroenteritis Loupingill virus Flavivirus, Flaviviridae Human, mammals, ticks Zoonosis,arthropod bite Encephalitis Lymphocytic Arenavirus, Arenaviridae Human,rodents Zoonosis, fomite Encephalitis choriomeningitis virus Machupovirus Arenavirus, Arenaviridae Human, monkeys, mouse Zoonosis, fomiteEncephalitis Mayaro virus Alphavirus, Togaviridae Human, mosquitoesZoonosis, arthropod bite Fever, joint pain MERS coronavirusBetacoronavirus, Coronaviridae Human, Tomb bat Zoonosis RespiratoryMeasles virus Morbilivirus, Paramyxoviridae Human Respirator, Fever,rash Mengo Cardiovirus, Picornaviridae Human, mouse, rabbit ZoonosisEncephalitis encephalomyocarditis virus Merkel cell polyomavirusPolyomavirus, Polyomaviridae Human — Merkel cell carcinoma Mokola virusLyssavirus, Rhabdoviridae Human, rodents, cat, dog shrew Zoonosis,animal bite Encephalitis Molluscum contagiosum virus Molluscipoxvirus,Poxviridae Human Contact Skin lesions Monkeypox virus OrthopoxvirusHuman, mouse, prairie dog Zoonosis, contact Skin lesions Mumps virusRubulavirus, Paramyxoviridae Human Respiratory, saliva Mumps Murrayvalley encephalitis virus Flavivirus Human, mosquitoes Zoonosis,arthropod bite Encephalitis New York virus Hantavirus, Bunyavirus Human,mouse Zoonosis, urine, saliva Hemorrhagic fever Nipah virus Henipavirus,Paramyxoviridae Human, bats Zoonosis, animal bite Encephalitis Norwalkvirus Norovirus, Caliciviridae Human Fecal-oral GastroenteritisO'nyong-nyong virus Alphavirus, Togaviridae Human, mosquitoes Zoonosis,arthropod bite Fever, joint pain Orf virus Parapoxvirus, PoxviridaeHuman, mammals Zoonosis, contact Skin lesions Oropouche virusOrthobunvavirus Human, wild animals(sloths) Zoonosis, arthropod biteFever, joint pain Pichinde virus Arenavirus, Arenaviridae Human, rat,guinea pig Zoonosis, fomite Hemorrhagic fever Poliovirus Enterovirus,Picornaviridae Human, mammals Fecal-oral Poliomyelitis Punta torophlebovirus Phlebovirus, Bunyaviridae Human, sandflies Zoonosis,arthropod bite Hemorrhagic fever Puumala virus Hantavirus, BunyavirusHuman, bank vole Zoonosis, urine, saliva Hemorrhagic fever Rabies virusLyssavirus, Rhabdoviridae Human, mammals Zoonosis, animal bite Fatalencephalitis Rift valley fever virus Phlebovirus, Bunvaviridae Human,mammals, Zoonosis, arthropod bite Hemorrhagic fever mosquitoes,sandflies Rosavirus A Rosavirus, Picornaviridae Human Ross river virusAlphavirus, Togaviridae Human, mosquitoes, Zoonosis, arthropod biteFever, joint pain marsupials Virus Genus, Family Host TransmissionDisease Rotavirus A Rotavirus, Rcoviridae Human Fecal-oralGastroenteritis Rotavirus B Rotavirus, Rcoviridae Human Fecal-oralGastroenteritis Rotavirus C Rotavirus, Rcoviridae Human Fecal-oralGastroenteritis Rubella virus Rubivirus, Togaviridae Human RespiratoryRubella Sagiyama virus Alphavirus, Togaviridae Human, horse, pig,Zoonosis, arthropod bite Fever, joint pain mosquitoes Salivirus ASalivirus, Picornaviridae Human Gastroenteritis Sandfly fever sicilianvirus Phlebovirus, Bunyaviridae Human, sandflies Zoonosis, arthropodbite Hemorrhagic fever Sapporo virus Sapovirus, Caliciviridae HumanFecal-oral Gastroenteritis Semliki forest virus Alphavirus, TogaviridaeHuman, birds, hedgehog, Zoonosis, arthropod bite Fever, joint painmosquitoes Seoul virus Hantavirus, Bunyavirus Human, rats Zoonosis,urine, saliva Hemorrhagic fever Simian foamy virus Spumavirus,Retroviridae Human, monkeys Zoonosis, contact None Simian virus 5Rubulavirus, Paramyxoviridae Human, dog Zoonosis, contact UndocumentedSindbis virus Alphavirus, Togaviridae Human, birds, mosquitoes Zoonosis,arthropod bite Pogosta_disease Fever, joint pain Southampton virusNorovirus, Caliciviridae Human Fecal-oral Gastroenteritis St. louisencephalitis virus Flavivirus, Flaviviridae Human, birds, mosquitoesZoonosis, arthropod bite Encephalitis Tick-borne powassan virusFlavivirus, Flaviviridae Human, ticks Zoonosis, arthropod biteEncephalitis Torque teno virus Alphatorquevirus Human Sexual, blood NoneToscana virus Phlebovirus, Bunyaviridae Human, mosquitoes Zoonosis,arthropod bite Hemorrhagic fever Uukuniemi virus Phleboviris,Bunyaviridae Human, ticks Zoonosis, arthropod bite Hemorrhagic feverVaccinia virus Orthopoxvirus, Poxviridae Human, mammals Contact NoneVaricella-zoster virus Varicellovirus, Herpesviridae Human Respiratory,contact Varicella Variola virus Orthopoxvirus, Poxviridae HumanRespiratory Variola Venezuelan equine Alphavirus, Togaviridae Human,rodents, mosquitoes Zoonosis, arthropod bite Fever, joint painencephalitis virus Vesicular stomatitis virus Vesiculovirus,Rhabdoviridae Human, cattle, horse, pig, flies Zoonosis, athropod biteEncephalitis Western equine Alphavirus, Togaviridae Human, vertebrates,Zoonosis, arthropod bite Fever, joint pain encephalitis virus mosquitoesWU polyomavirus Polyomavirus, Polyomaviridae Human Respiratory fluids orurine None West Nile virus Flavivirus, Flaviviridae Human, birds, ticks,Zoonosis, arthropod bite Hemorrhagic fever mosquitoes Yaba monkey tumorvirus Orthopoxvirus, Poxviridae Human, monkeys Zoonosis, contact NoneYaba-like disease virus Orthopoxvirus, Poxviridae Human, monkeysZoonosis, contact None Yellow fever virus Flavivirus, FlaviviridaeHuman, monkeys, mosquitoes Zoonosis, arthropod bite Hemorrhagic feverZika virus Flavivirus, Flaviviridae Human, monkeys, mosauitoes Zoonosis,arthropod bite Fever, joint pain, rash

In certain embodiments, the virus is a virus listed in Table 2 below.The type of delivery will be dependent upon the tissue/cell tropism ofthe RNA (or DNA) virus of interest. Accordingly, in certain embodiments,the virus is a virus listed in Table 2 below and the delivery vehicle issuitable for delivery to the cells or tissues or organs (correspondingto the indicated virus) listed in Table 2 below.

TABLE 2 Virus Tissue/cell type Citation Lassa virus DCs, vascularendothelial Kunz, S. et. al. 2005. Journal of cells Virology Ebola virusNumerous (DCs, Martines, R.B. et. al. 2015. macrophages, hepatocytes,Journal of Pathology. etc.) SARS-CoV Lung To, KF. et. al. 2004. Journalof Pathology. Zika Numerous (bodily fluids, Miner, J.J. & Diamond, M.S.placenta, brain, etc.) 2017. Cell Host & Microbe. Dengue Numerous (DCs,Flipse, J. et. al. 2016. Journal macrophages, liver, etc.) of GeneralVirology. Chikungunya Numerous (immune cells, Schwartz, O. & Albert,M.L. liver, central nervous 2010. Nature Reviews. system, etc.)Influenza Lung epithelial cells or Medina, R.A. & Garcia-Sastremacrophages A. 2011 Nature Reviews. HIV T cells, macrophages Weiss, R.A.2002. IUBMB Life. Rotavirus Intestine Lopez, S & Arias, C.F. 2006. CTMIHerpes Epithelial cells, neuronal Schelhaas, M. et. al. 2003. Simplexcells Journal of General Virology. (HSV-1) HCV Liver Ding, Q, et. al.2014. Cell Host & Microbe. HBV Liver Schieck, A. et. al. 2013.Hepatology.

In certain embodiments, the virus is a virus listed in Table 3 below.

TABLE 3 List of Viruses with FDA-Approved Vaccines (15-16): 1.Adenovirus 2. Hepatitis A 3. Hepatitis B 4. Human Papillomavirus (HPV)5. Influenza 6. Japanese Encephalitis Virus 7. Measles 8. Mumps 9. Polio10. Rabies 11. Rotavirus 12. Rubella 13. Shingles/Zoster (HSV) 14.Smallpox* 15. Varicella (Chicken Pox) 16. Yellow Fever List of Viruseswith FDA-Approved Antiviral Drugs (9): 1. Cytomegalovirus 2. HumanImmunodeficiency Virus (HIV) 3. Hepatitis B 4. Hepatitis C 5. Influenza6. Respiratory Syncytial Virus 7. Human Papillomavirus (HPV) 8. HerpesSimplex Virus (Shingles) 9. Varicella Zoster Virus (Chicken pox) List ofViruses with FDA-Approved Nucleic Acid Diagnostics (11): 1. Adenovirus2. Cytomegalovirus 3. Dengue 4. Enterovirus 5. Herpes Simplex Virus 6.Hepatitis B 7. Hepatitis C 8. Human Metapneumovirus 9. HumanPapillomavirus 10. Influenza 11. Respiratory Syncytial Virus

In certain example embodiments, the virus may be a plant virus selectedfrom the group comprising Tobacco mosaic virus (TMV), Tomato spottedwilt virus (TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY),the RT virus Cauliflower mosaic virus (CaMV), Plum pox virus (PPV),Brome mosaic virus (BMV), Potato virus X (PVX), Citrus tristeza virus(CTV), Barley yellow dwarf virus (BYDV), Potato leafroll virus (PLRV),Tomato bushy stunt virus (TBSV), rice tungro spherical virus (RTSV),rice yellow mottle virus (RYMV), rice hoja blanca virus (RHBV), maizerayado fino virus (MRFV), maize dwarf mosaic virus (MDMV), sugarcanemosaic virus (SCMV), Sweet potato feathery mottle virus (SPFMV), sweetpotato sunken vein closterovirus (SPSVV), Grapevine fanleaf virus(GFLV), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevinefleck virus (GFkV), Grapevine leafroll-associated virus-1, -2, and -3,(GLRaV-1, -2, and -3), Arabis mosaic virus (ArMV), or Rupestris stempitting-associated virus (RSPaV).

In a preferred embodiment, the target RNA molecule is part of saidpathogen or transcribed from a DNA molecule of said pathogen. Forexample, the target sequence may be comprised in the genome of an RNAvirus. It is further preferred that CRISPR effector protein hydrolyzessaid target RNA molecule of said pathogen in said plant if said pathogeninfects or has infected said plant. It is thus preferred that the CRISPRsystem is capable of cleaving the target RNA molecule from the plantpathogen both when the CRISPR system (or parts needed for itscompletion) is applied therapeutically, i.e. after infection hasoccurred or prophylactically, i.e. before infection has occurred.

In certain example embodiments, the virus may be a retrovirus. Exampleretroviruses that may be detected using the embodiments disclosed hereininclude one or more of or any combination of viruses of the GenusAlpharetrovirus, Betaretrovirus, Gammaretrovirus, Deltaretrovirus,Epsilonretrovirus, Lentivirus, Spumavirus, or the Family Metaviridae,Pseudoviridae, and Retroviridae (including HIV), Hepadnaviridae(including Hepatitis B virus), and Caulimoviridae (including Cauliflowermosaic virus).

In certain example embodiments, the virus is a DNA virus. Example DNAviruses that may be detected using the embodiments disclosed hereininclude one or more of (or any combination of) viruses from the FamilyMyoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae(including human herpes virus, and Varicella Zozter virus),Malocoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae,Ampullaviridae, Ascoviridae, Asfarviridae (including African swine fevervirus), Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae,Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae,Iridoviridae, Maseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae,Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae,Polydnaviruses, Polyomaviridae (including Simian virus 40, JC virus, BKvirus), Poxviridae (including Cowpox and smallpox), Sphaerolipoviridae,Tectiviridae, Turriviridae, Dinodnavirus, Salterprovirus, Rhizidovirus,among others.

In certain embodiments, the virus is a drug resistant virus. By means ofexample, and without limitation, the virus may be a ribavirin resistantvirus. Ribavirin is a very effective antiviral that hits a number of RNAviruses. Below are a few important viruses that have evolved ribavirinresistance. Foot and Mouth Disease Virus: doi:10.1128/JVI.03594-13.Polio virus: http://www.pnas.org/content/100/12/7289.full.pdf. HepatitisC Virus: http://jvi.asm.org/content/79/4/2346.full. A number of otherpersistent RNA viruses, such as hepatitis and HIV, have evolvedresistance to existing antiviral drugs. Hepatitis B Virus (lamivudine,tenofovir, entecavir): doi:10.1002/hep.22900. Hepatitis C Virus(Telaprevir, BILN2061, ITMN-191, SCH6, Boceprevir, AG-021541, ACH-806):doi:10.1002/hep.22549. HIV has many drug resistant mutations, seehttps://hivdb.stanford.edu/ for more information. Aside from drugresistance, there are a number of clinically relevant mutations thatcould be targeted with the CRISPR systems according to the invention asdescribed herein. For instance, persistent versus acute infection inLCMV: doi: 10.1073/pnas.1019304108; or increased infectivity of Ebola:http://doi.org/10.1016/j.cell.2016.10.014 andhttp://doi.org/10.1016/j.cell.2016.10.013.

General Provisions

In an aspect, the invention provides a nucleic acid binding system, i.e.a CRISPR system or CRISPR/Cas system, more in particular a Class 2 typeVI Crispr system. The nucleic acid binding system as described hereinessentially comprises a CRISPR effector protein and a guide RNA.

In embodiments of the invention a guide RNA comprises a guide sequenceand a direct repeat sequence. In general, a guide sequence (also calledspacer sequence) is any polynucleotide sequence having sufficientcomplementarity with a target polynucleotide sequence to hybridize withthe target sequence and direct sequence-specific binding of a CRISPRcomplex to the target sequence. In some embodiments, the degree ofcomplementarity between a guide sequence and its corresponding targetsequence, when optimally aligned using a suitable alignment algorithm,is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%,99%, or more. Optimal alignment may be determined with the use of anysuitable algorithm for aligning sequences, non-limiting example of whichinclude the Smith-Waterman algorithm, the Needleman-Wunsch algorithm,algorithms based on the Burrows-Wheeler Transform (e.g., the BurrowsWheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (NovocraftTechnologies; available at www.novocraft.com), ELAND (Illumina, SanDiego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq(available at maq.sourceforge.net). In some embodiments, a guidesequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75,or more nucleotides in length. In some embodiments, a guide sequence isless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length. Preferably the guide sequence is 10-30nucleotides long. The ability of a guide sequence to directsequence-specific binding of a CRISPR complex to a target sequence maybe assessed by any suitable assay. For example, the components of aCRISPR system sufficient to form a CRISPR complex, including the guidesequence to be tested, may be provided to a host cell having thecorresponding target sequence, such as by transfection with vectorsencoding the components of the CRISPR sequence, followed by anassessment of preferential cleavage within the target sequence, such asby Surveyor assay as described herein. Similarly, cleavage of a targetpolynucleotide sequence may be evaluated in a test tube by providing thetarget sequence, components of a CRISPR complex, including the guidesequence to be tested and a control guide sequence different from thetest guide sequence, and comparing binding or rate of cleavage at thetarget sequence between the test and control guide sequence reactions.Other assays are possible, and will occur to those skilled in the art. Aguide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a genome of acell. Exemplary target sequences include those that are unique in thetarget genome.

In general, and throughout this specification, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses). Viralvectors also include polynucleotides carried by a virus for transfectioninto a host cell. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) areintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors.” Vectors for and that result inexpression in a eukaryotic cell can be referred to herein as “eukaryoticexpression vectors.” Common expression vectors of utility in recombinantDNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

As used herein, the term “crRNA” or “guide RNA” or “single guide RNA” or“sgRNA” or “one or more nucleic acid components” of a Type V or Type VICRISPR-Cas locus effector protein comprises any polynucleotide sequencehaving sufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a nucleic acid-targeting complex to thetarget nucleic acid sequence. In some embodiments, the degree ofcomplementarity, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X,BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).The ability of a guide sequence (within a nucleic acid-targeting guideRNA) to direct sequence-specific binding of a nucleic acid-targetingcomplex to a target nucleic acid sequence may be assessed by anysuitable assay. For example, the components of a nucleic acid-targetingCRISPR system sufficient to form a nucleic acid-targeting complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target nucleic acid sequence, such as bytransfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget nucleic acid sequence may be evaluated in a test tube byproviding the target nucleic acid sequence, components of a nucleicacid-targeting complex, including the guide sequence to be tested and acontrol guide sequence different from the test guide sequence, andcomparing binding or rate of cleavage at the target sequence between thetest and control guide sequence reactions. Other assays are possible,and will occur to those skilled in the art. A guide sequence, and hencea nucleic acid-targeting guide RNA may be selected to target any targetnucleic acid sequence. The target sequence may be DNA. The targetsequence may be any RNA sequence. In some embodiments, the targetsequence may be a sequence within a RNA molecule selected from the groupconsisting of messenger RNA (mRNA), pre-mRNA, ribosomaal RNA (rRNA),transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA),small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double strandedRNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA (lncRNA), andsmall cytoplasmatic RNA (scRNA). In some preferred embodiments, thetarget sequence may be a sequence within a RNA molecule selected fromthe group consisting of mRNA, pre-mRNA, and rRNA. In some preferredembodiments, the target sequence may be a sequence within a RNA moleculeselected from the group consisting of ncRNA, and lncRNA. In some morepreferred embodiments, the target sequence may be a sequence within anmRNA molecule or a pre-mRNA molecule.

In some embodiments, a nucleic acid-targeting guide RNA is selected toreduce the degree secondary structure within the RNA-targeting guideRNA. In some embodiments, about or less than about 75%, 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleicacid-targeting guide RNA participate in self-complementary base pairingwhen optimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carrand GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

These RNA structure prediction algorithms may also be used to predictthe structure of the target RNA. As target RNA structure may have aninfluence of guide RNA binding efficiency or CRISPR system cleavageefficiency, prediction of the target RNA structure allows rationaldesign of guide RNAs, such that for instance guide RNAs may be chosen tobind less structured areas of the target RNA, in order to improve forinstance accessability. Accordingly, in certain embodiments, the one ormore guide RNA as described herein bind less structured or unstructuredareas of the target RNA. In certain embodiments, the guide RNA binds toaccessible areas of the target RNA.

In certain embodiments, a guide RNA or crRNA may comprise, consistessentially of, or consist of a direct repeat (DR) sequence and a guidesequence or spacer sequence. In certain embodiments, the guide RNA orcrRNA may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.In certain embodiments, the direct repeat sequence may be locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In otherembodiments, the direct repeat sequence may be located downstream (i.e.,3′) from the guide sequence or spacer sequence.

In certain embodiments, the crRNA comprises a stem loop, preferably asingle stem loop. In certain embodiments, the direct repeat sequenceforms a stem loop, preferably a single stem loop.

In certain embodiments, the spacer length of the guide RNA is from 15 to35 nt. In certain embodiments, the spacer length of the guide RNA is atleast 15 nucleotides, preferably at least 18 nt, such at at least 19,20, 21, 22, or more nt. In certain embodiments, the spacer length isfrom 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17,18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26,or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt,e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.

Also described is a challenge experiment to verify the RNA targeting andcleaving capability of a CRISPR effector. This experiment closelyparallels similar work in E. coli for the heterologous expression ofStCas9 (Sapranauskas, R. et al. Nucleic Acids Res 39, 9275-9282 (2011)).A plasmid containing both a PAM and a resistance gene are introducedinto the heterologous E. coli, and then plate on the correspondingantibiotic. If there is RNA cleavage of the plasmid transcribedresistance gene, no viable colonies are observed.

In further detail, the assay is as follows for a DNA target, but may beadapted accordingly for an RNA target. Two E. coli strains are used inthis assay. One carries a plasmid that encodes the endogenous effectorprotein locus from the bacterial strain. The other strain carries anempty plasmid (e.g. pACYC184, control strain). All possible 7 or 8 bpPAM sequences are presented on an antibiotic resistance plasmid (pUC19with ampicillin resistance gene). The PAM is located next to thesequence of proto-spacer 1 (the DNA target to the first spacer in theendogenous effector protein locus). Two PAM libraries were cloned. Onehas a 8 random bp 5′ of the proto-spacer (e.g. total of 65536 differentPAM sequences=complexity). The other library has 7 random bp 3′ of theproto-spacer (e.g. total complexity is 16384 different PAMs). Bothlibraries were cloned to have in average 500 plasmids per possible PAM.Test strain and control strain were transformed with 5′PAM and 3′PAMlibrary in separate transformations and transformed cells were platedseparately on ampicillin plates. Recognition and subsequentcutting/interference with the plasmid renders a cell vulnerable toampicillin and prevents growth. Approximately 12 h after transformation,all colonies formed by the test and control strains where harvested andplasmid DNA was isolated. Plasmid DNA was used as template for PCRamplification and subsequent deep sequencing. Representation of all PAMsin the untransfomed libraries showed the expected representation of PAMsin transformed cells. Representation of all PAMs found in controlstrains showed the actual representation. Representation of all PAMs intest strain showed which PAMs are not recognized by the enzyme andcomparison to the control strain allows extracting the sequence of thedepleted PAM. It will be understood that the above allows identificationof PAM (or PFS) sequences (5′ and/or 3′) for any given CRISPR effectororthologue.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of nucleic acid-targeting guide RNAdelivered. Optimal concentrations of nucleic acid-targeting guide RNAcan be determined by testing different concentrations in a cellular ornon-human eukaryote animal model and using deep sequencing the analyzethe extent of modification at potential off-target genomic loci. Theconcentration that gives the highest level of on-target modificationwhile minimizing the level of off-target modification should be chosenfor in vivo delivery. The nucleic acid-targeting system is derivedadvantageously from a Type VI CRISPR system. In some embodiments, one ormore elements of a nucleic acid-targeting system is derived from aparticular organism comprising an endogenous RNA-targeting system. Inparticular embodiments, the Type VI RNA-targeting Cas enzyme is Cas 13a(C2c2) or Cas13b. In embodiments, the Type VI CRISPR effector proteinsuch as C2c2 as referred to herein also encompasses a homologue or anorthologue of a Type VI protein. The terms “orthologue” (also referredto as “ortholog” herein) and “homologue” (also referred to as “homolog”herein) are well known in the art. By means of further guidance, a“homologue” of a protein as used herein is a protein of the same specieswhich performs the same or a similar function as the protein it is ahomologue of. Homologous proteins may but need not be structurallyrelated, or are only partially structurally related. An “orthologue” ofa protein as used herein is a protein of a different species whichperforms the same or a similar function as the protein it is anorthologue of. Orthologous proteins may but need not be structurallyrelated, or are only partially structurally related. In particularembodiments, the homologue or orthologue of a Type VI protein such asC2c2 as referred to herein has a sequence homology or identity of atleast 80%, more preferably at least 85%, even more preferably at least90%, such as for instance at least 95% with a Type VI protein such asC2c2. In further embodiments, the homologue or orthologue of a Type VIprotein such as C2c2 as referred to herein has a sequence identity of atleast 80%, more preferably at least 85%, even more preferably at least90%, such as for instance at least 95% with the wild type Type VIprotein such as C2c2.

In an embodiment, the Type VI RNA-targeting Cas protein may be a C2c2ortholog of an organism of a genus which includes but is not limited toLeptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema,Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma,Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma and Campylobacter. Species of organism ofsuch a genus can be as otherwise herein discussed.

Some methods of identifying orthologs of CRISPR-Cas system enzymes mayinvolve identifying tracr sequences in genomes of interest.Identification of tracr sequences may relate to the following steps:Search for the direct repeats or tracr mate sequences in a database toidentify a CRISPR region comprising a CRISPR enzyme. Search forhomologous sequences in the CRISPR region flanking the CRISPR enzyme inboth the sense and antisense directions. Look for transcriptionalterminators and secondary structures. Identify any sequence that is nota direct repeat or a tracr mate sequence but has more than 50% identityto the direct repeat or tracr mate sequence as a potential tracrsequence. Take the potential tracr sequence and analyze fortranscriptional terminator sequences associated therewith.

It will be appreciated that any of the functionalities described hereinmay be engineered into CRISPR enzymes from other orthologs, includingchimeric enzymes comprising fragments from multiple orthologs. Examplesof such orthologs are described elsewhere herein. Thus, chimeric enzymesmay comprise fragments of CRISPR enzyme orthologs of an organism whichincludes but is not limited to Leptotrichia, Listeria, Corynebacter,Sutterella, Legionella, Treponema, Filifactor, Eubacterium,Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola,Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter,Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor,Mycoplasma and Campylobacter. A chimeric enzyme can comprise a firstfragment and a second fragment, and the fragments can be of CRISPRenzyme orthologs of organisms of genuses herein mentioned or of speciesherein mentioned; advantageously the fragments are from CRISPR enzymeorthologs of different species.

In embodiments, the Type VI RNA-targeting effector protein, inparticular the C2c2 protein as referred to herein also encompasses afunctional variant of C2c2 or a homologue or an orthologue thereof. A“functional variant” of a protein as used herein refers to a variant ofsuch protein which retains at least partially the activity of thatprotein. Functional variants may include mutants (which may beinsertion, deletion, or replacement mutants), including polymorphs, etc.Also included within functional variants are fusion products of suchprotein with another, usually unrelated, nucleic acid, protein,polypeptide or peptide. Functional variants may be naturally occurringor may be man-made. Advantageous embodiments can involve engineered ornon-naturally occurring Type VI RNA-targeting effector protein.

In an embodiment, nucleic acid molecule(s) encoding the Type VIRNA-targeting effector protein, in particular C2c2 or an ortholog orhomolog thereof, may be codon-optimized for expression in an eukaryoticcell. A eukaryote can be as herein discussed. Nucleic acid molecule(s)can be engineered or non-naturally occurring.

In an embodiment, the Type VI RNA-targeting effector protein, inparticular C2c2 or an ortholog or homolog thereof, may comprise one ormore mutations (and hence nucleic acid molecule(s) coding for same mayhave mutation(s). The mutations may be artificially introduced mutationsand may include but are not limited to one or more mutations in acatalytic domain. Examples of catalytic domains with reference to a Cas9enzyme may include but are not limited to RuvC I, RuvC II, RuvC III andHNH domains.

In an embodiment, the Type VI protein such as C2c2 or an ortholog orhomolog thereof, may comprise one or more mutations. The mutations maybe artificially introduced mutations and may include but are not limitedto one or more mutations in a catalytic domain. Examples of catalyticdomains with reference to a Cas enzyme may include but are not limitedto HEPN domains.

In an embodiment, the Type VI protein such as C2c2 or an ortholog orhomolog thereof, may be used as a generic nucleic acid binding proteinwith fusion to or being operably linked to a functional domain.Exemplary functional domains may include but are not limited totranslational initiator, translational activator, translationalrepressor, nucleases, in particular ribonucleases, a spliceosome, beads,a light inducible/controllable domain or a chemicallyinducible/controllable domain.

In some embodiments, the unmodified nucleic acid-targeting effectorprotein may have cleavage activity. In some embodiments, theRNA-targeting effector protein may direct cleavage of one or bothnucleic acid strands at the location of or near a target sequence, suchas within the target sequence and/or within the complement of the targetsequence or at sequences associated with the target sequence. In someembodiments, the nucleic acid-targeting Cas protein may direct cleavageof one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 50, 100, 200, 500, or more base pairs from the first or lastnucleotide of a target sequence. In some embodiments, a vector encodes anucleic acid-targeting Cas protein that may be mutated with respect to acorresponding wild-type enzyme such that the mutated nucleicacid-targeting Cas protein lacks the ability to cleave RNA strands of atarget polynucleotide containing a target sequence. As a furtherexample, two or more catalytic domains of Cas (e.g. HEPN domain) may bemutated to produce a mutated Cas substantially lacking all RNA cleavageactivity. In some embodiments, a nucleic acid-targeting effector proteinmay be considered to substantially lack all RNA cleavage activity whenthe RNA cleavage activity of the mutated enzyme is about no more than25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the nucleic acid cleavageactivity of the non-mutated form of the enzyme; an example can be whenthe nucleic acid cleavage activity of the mutated form is nil ornegligible as compared with the non-mutated form. An effector proteinmay be identified with reference to the general class of enzymes thatshare homology to the biggest nuclease with multiple nuclease domainsfrom the Type VI CRISPR system. Most preferably, the effector protein isa Type VI protein such as C2c2. By derived, Applicants mean that thederived enzyme is largely based, in the sense of having a high degree ofsequence homology with, a wildtype enzyme, but that it has been mutated(modified) in some way as known in the art or as described herein.

Again, it will be appreciated that the terms Cas and CRISPR enzyme andCRISPR protein and Cas protein are generally used interchangeably and atall points of reference herein refer by analogy to novel CRISPR effectorproteins further described in this application, unless otherwiseapparent, such as by specific reference to Cas9. As mentioned above,many of the residue numberings used herein refer to the effector proteinfrom the Type VI CRISPR locus. However, it will be appreciated that thisinvention includes many more effector proteins from other species ofmicrobes. In certain embodiments, Cas may be constitutively present orinducibly present or conditionally present or administered or delivered.Cas optimization may be used to enhance function or to develop newfunctions, one can generate chimeric Cas proteins. And Cas may be usedas a generic nucleic acid binding protein.

Typically, in the context of an endogenous nucleic acid-targetingsystem, formation of a nucleic acid-targeting complex (comprising aguide RNA hybridized to a target sequence and complexed with one or morenucleic acid-targeting effector proteins) results in cleavage of one orboth DNA or RNA strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 50, 100, 200, 500, or more base pairs from) the targetsequence. As used herein the term “sequence(s) associated with a targetlocus of interest” refers to sequences near the vicinity of the targetsequence (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200,500, or more base pairs from the target sequence, wherein the targetsequence is comprised within a target locus of interest).

An example of a codon optimized sequence, is in this instance a sequenceoptimized for expression in a eukaryote, e.g., humans (i.e. beingoptimized for expression in humans), or for another eukaryote, animal ormammal as herein discussed; see, e.g., SaCas9 human codon optimizedsequence in WO 2014/093622 (PCT/US2013/074667) as an example of a codonoptimized sequence (from knowledge in the art and this disclosure, codonoptimizing coding nucleic acid molecule(s), especially as to effectorprotein (e.g., C2c2) is within the ambit of the skilled artisan). Whilstthis is preferred, it will be appreciated that other examples arepossible and codon optimization for a host species other than human, orfor codon optimization for specific organs is known. In someembodiments, an enzyme coding sequence encoding a DNA/RNA-targeting Casprotein is codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a mammal, including but not limited tohuman, or non-human eukaryote or animal or mammal as herein discussed,e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal orprimate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. In general,codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g., about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at www.kazusa.orjp/codon/ and these tables canbe adapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codonoptimizing a particular sequence for expression in a particular hostcell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), arealso available. In some embodiments, one or more codons (e.g., 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encodinga DNA/RNA-targeting Cas protein corresponds to the most frequently usedcodon for a particular amino acid.

As used herein, the term “viral load” refers to viral burden, viraltitre or viral titer, and is a numerical expression of the quantity ofvirus in a given volume, determined as viral particles, or infectiousparticles per ml.

Crispr Effector Proteins

In general, a CRISPR-Cas or CRISPR system as used in herein and indocuments, such as WO 2014/093622 (PCT/US2013/074667), referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, such as Cas13a, Cas13b,or Cas 13c in certain embodiments of the invention, a tracr(trans-activating CRISPR) sequence (e.g. tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNAand transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). When the CRISPR protein is a C2c2 protein, a tracrRNA is notrequired. C2c2 has been described in Abudayyeh et al. (2016) “C2c2 is asingle-component programmable RNA-guided RNA-targeting CRISPR effector”;Science; DOI: 10.1126/science.aaf5573; and Shmakov et al. (2015)“Discovery and Functional Characterization of Diverse Class 2 CRISPR-CasSystems”, Molecular Cell, DOI: dx.doi.org/10.1016/j.molcel.2015.10.008;which are incorporated herein in their entirety by reference. Cas13b hasbeen described in Smargon et al. (2017) “Cas13b Is a Type VI-BCRISPR-Associated RNA-Guided RNases Differentially Regulated byAccessory Proteins Csx27 and Csx28,” Molecular Cell. 65, 1-13;dx.doi.org/10.1016/j.molcel.2016.12.023., which is incorporated hereinin its entirety by reference.

In certain embodiments, a protospacer adjacent motif (PAM) or PAM-likemotif or protospacer flanking sequence (PFS) directs binding of theeffector protein complex as disclosed herein to the target locus ofinterest. In some embodiments, the PAM may be a 5′ PAM (i.e., locatedupstream of the 5′ end of the protospacer). In other embodiments, thePAM may be a 3′ PAM (i.e., located downstream of the 5′ end of theprotospacer). The term “PAM” may be used interchangeably with the term“PFS” or “protospacer flanking site” or “protospacer flanking sequence”.

In a preferred embodiment, the CRISPR effector protein may recognize a3′ PAM. In certain embodiments, the CRISPR effector protein mayrecognize a 3′ PAM which is 5′H, wherein H is A, C or U. In certainembodiments, the effector protein may be Leptotrichia shahii C2c2p, morepreferably Leptotrichia shahii DSM 19757 C2c2, and the 3′ PAM is a 5′ H.

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise RNA polynucleotides. The term “target RNA” refersto a RNA polynucleotide being or comprising the target sequence. Inother words, the target RNA may be a RNA polynucleotide or a part of aRNA polynucleotide to which a part of the gRNA, i.e. the guide sequence,is designed to have complementarity and to which the effector functionmediated by the complex comprising CRISPR effector protein and a gRNA isto be directed. In some embodiments, a target sequence is located in thenucleus or cytoplasm of a cell.

The nucleic acid molecule encoding a CRISPR effector protein, inparticular C2c2 or Cas13b, is advantageously codon optimized CRISPReffector protein. An example of a codon optimized sequence, is in thisinstance a sequence optimized for expression in eukaryote, e.g., humans(i.e. being optimized for expression in humans), or for anothereukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 humancodon optimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilstthis is preferred, it will be appreciated that other examples arepossible and codon optimization for a host species other than human, orfor codon optimization for specific organs is known. In someembodiments, an enzyme coding sequence encoding a CRISPR effectorprotein is a codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a plant or a mammal, including but notlimited to human, or non-human eukaryote or animal or mammal as hereindiscussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammalor primate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. In general,codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g. about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at kazusa.orjp/codon/ and these tables can beadapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codonoptimizing a particular sequence for expression in a particular hostcell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), arealso available. In some embodiments, one or more codons (e.g. 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encodinga Cas correspond to the most frequently used codon for a particularamino acid.

In certain embodiments, the methods as described herein may compriseproviding a Cas transgenic cell, tissue, organ, or organism, inparticular a C2c2 or Cas13b transgenic cell, tissue, organ, or organism,in which one or more guide RNAs or nucleic acids encoding one or moreguide RNAs operably connected in the cell with a regulatory elementcomprising a promoter of one or more gene of interest are provided orintroduced. As used herein, the term “Cas transgenic cell” refers to acell, such as a eukaryotic cell, in which a Cas gene, such as a C2c2 arCAS13b gene, has been genomically integrated. The nature, type, ororigin of the cell are not particularly limiting according to thepresent invention. Also the way the Cas transgene is introduced in thecell may vary and can be any method as is known in the art. In certainembodiments, the Cas transgenic cell is obtained by introducing the Castransgene in an isolated cell. In certain other embodiments, the Castransgenic cell is obtained by isolating cells from a Cas transgenicorganism. By means of example, and without limitation, the Castransgenic cell as referred to herein may be derived from a Castransgenic eukaryote, such as a Cas knock-in eukaryote. Reference ismade to WO 2014/093622 (PCT/US13/74667), incorporated herein byreference. Methods of US Patent Publication Nos. 20120017290 and20110265198 assigned to Sangamo BioSciences, Inc. directed to targetingthe Rosa locus may be modified to utilize the CRISPR Cas system of thepresent invention. Methods of US Patent Publication No. 20130236946assigned to Cellectis directed to targeting the Rosa locus may also bemodified to utilize the CRISPR Cas system of the present invention. Bymeans of further example reference is made to Platt et. al. (Cell;159(2):440-455 (2014)), describing a Cas9 knock-in mouse, which isincorporated herein by reference. The Cas transgene can further comprisea Lox-Stop-polyA-Lox (LSL) cassette thereby rendering Cas expressioninducible by Cre recombinase. Alternatively, the Cas transgenic cell maybe obtained by introducing the Cas transgene in an isolated cell.Delivery systems for transgenes are well known in the art. By means ofexample, the Cas transgene may be delivered in for instance eukaryoticcell by means of vector (e.g., AAV, adenovirus, lentivirus) and/orparticle and/or nanoparticle delivery, as also described hereinelsewhere.

It will be understood by the skilled person that the cell, such as theCas transgenic cell, as referred to herein may comprise further genomicalterations besides having an integrated Cas gene or the mutationsarising from the sequence specific action of Cas when complexed with RNAcapable of guiding Cas to a target locus.

In one example embodiment, the effector protein comprise one or moreHEPN domains comprising a RxxxxH motif sequence. The RxxxxH motifsequence can be, without limitation, from a HEPN domain described hereinor a HEPN domain known in the art. RxxxxH motif sequences furtherinclude motif sequences created by combining portions of two or moreHEPN domains. As noted, consensus sequences can be derived from thesequences of the orthologs disclosed in U.S. Provisional PatentApplication 62/432,240 entitled “Novel CRISRP Enzymes and Systems” andU.S. Provisional patent application entitled “Novel CRISPR Enzymes andSystems” filed on Mar. 15, 2017.

In an embodiment of the invention, a HEPN domain comprises at least oneRxxxxH motif comprising the sequence of R{N/H/K}X1X2X3H. In anembodiment of the invention, a HEPN domain comprises a RxxxxH motifcomprising the sequence of R{N/H}X1X2X3H. In an embodiment of theinvention, a HEPN domain comprises the sequence of R{N/K}X1X2X3H. Incertain embodiments, X1 is R, S, D, E, Q, N, G, Y, or H. In certainembodiments, X2 is I, S, T, V, or L. In certain embodiments, X3 is L, F,N, Y, V, I, S, D, E, or A.

Additional effectors for use according to the invention can beidentified by their proximity to cas1 genes, for example, though notlimited to, within the region 20 kb from the start of the cas1 gene and20 kb from the end of the cas1 gene. In certain embodiments, theeffector protein comprises at least one HEPN domain and at least 500amino acids, and wherein the CRISPR effector protein is naturallypresent in a prokaryotic genome within 20 kb upstream or downstream of aCas gene or a CRISPR array. In certain example embodiments, the CRISPReffector protein is naturally present in a prokaryotic genome within 20kb upstream or downstream of a Cas 1 gene. The terms “orthologue” (alsoreferred to as “ortholog” herein) and “homologue” (also referred to as“homolog” herein) are well known in the art. By means of furtherguidance, a “homologue” of a protein as used herein is a protein of thesame species which performs the same or a similar function as theprotein it is a homologue of. Homologous proteins may but need not bestructurally related, or are only partially structurally related. An“orthologue” of a protein as used herein is a protein of a differentspecies which performs the same or a similar function as the protein itis an orthologue of. Orthologous proteins may but need not bestructurally related, or are only partially structurally related

In particular embodiments, the Type VI RNA-targeting Cas enzyme is C2c2.In other example embodiments, the Type VI RNA-targeting Cas enzyme isCas 13b. In particular embodiments, the homologue or orthologue of aType VI protein such as C2c2 as referred to herein has a sequencehomology or identity of at least 30%, or at least 40%, or at least 50%,or at least 60%, or at least 70%, or at least 80%, more preferably atleast 85%, even more preferably at least 90%, such as for instance atleast 95% with a Type VI protein such as C2c2 (e.g., based on thewild-type sequence of any of Leptotrichia shahii C2c2, Lachnospiraceaebacterium MA2020 C2c2, Lachnospiraceae bacterium NK4A179 C2c2,Clostridium aminophilum (DSM 10710) C2c2, Carnobacterium gallinarum (DSM4847) C2c2, Paludibacter propionicigenes (WB4) C2c2, Listeriaweihenstephanensis (FSL R9-0317) C2c2, Listeriaceae bacterium (FSLM6-0635) C2c2, Listeria newyorkensis (FSL M6-0635) C2c2, Leptotrichiawadei (F0279) C2c2, Rhodobacter capsulatus (SB 1003) C2c2, Rhodobactercapsulatus (R121) C2c2, Rhodobacter capsulatus (DE442) C2c2,Leptotrichia wadei (Lw2) C2c2, or Listeria seeligeri C2c2). In furtherembodiments, the homologue or orthologue of a Type VI protein such asC2c2 as referred to herein has a sequence identity of at least 30%, orat least 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, more preferably at least 85%, even more preferably at least90%, such as for instance at least 95% with the wild type C2c2 (e.g.,based on the wild-type sequence of any of Leptotrichia shahii C2c2,Lachnospiraceae bacterium MA2020 C2c2, Lachnospiraceae bacterium NK4A179C2c2, Clostridium aminophilum (DSM 10710) C2c2, Carnobacteriumgallinarum (DSM 4847) C2c2, Paludibacter propionicigenes (WB4) C2c2,Listeria weihenstephanensis (FSL R9-0317) C2c2, Listeriaceae bacterium(FSL M6-0635) C2c2, Listeria newyorkensis (FSL M6-0635) C2c2,Leptotrichia wadei (F0279) C2c2, Rhodobacter capsulatus (SB 1003) C2c2,Rhodobacter capsulatus (R121) C2c2, Rhodobacter capsulatus (DE442) C2c2,Leptotrichia wadei (Lw2) C2c2, or Listeria seeligeri C2c2).

In certain other example embodiments, the CRISPR system the effectorprotein is a C2c2 nuclease. The activity of C2c2 may depend on thepresence of two HEPN domains. These have been shown to be RNase domains,i.e. nuclease (in particular an endonuclease) cutting RNA. C2c2 HEPN mayalso target DNA, or potentially DNA and/or RNA. On the basis that theHEPN domains of C2c2 are at least capable of binding to and, in theirwild-type form, cutting RNA, then it is preferred that the C2c2 effectorprotein has RNase function. Regarding C2c2 CRISPR systems, reference ismade to U.S. Provisional 62/351,662 filed on Jun. 17, 2016 and U.S.Provisional 62/376,377 filed on Aug. 17, 2016. Reference is also made toU.S. Provisional 62/351,803 filed on Jun. 17, 2016. Reference is alsomade to U.S. Provisional entitled “Novel Crispr Enzymes and Systems”filed Dec. 8, 2016 bearing Broad Institute No. 10035.PA4 and AttorneyDocket No. 47627.03.2133. Reference is further made to East-Seletsky etal. “Two distinct RNase activities of CRISPR-C2c2 enable guide-RNAprocessing and RNA detection” Nature doi:10/1038/nature19802 andAbudayyeh et al. “C2c2 is a single-component programmable RNA-guided RNAtargeting CRISPR effector” bioRxiv doi: 10.1101/054742.

RNase function in CRISPR systems is known, for example mRNA targetinghas been reported for certain type III CRISPR-Cas systems (Hale et al.,2014, Genes Dev, vol. 28, 2432-2443; Hale et al., 2009, Cell, vol. 139,945-956; Peng et al., 2015, Nucleic acids research, vol. 43, 406-417)and provides significant advantages. In the Staphylococcus epidermistype III-A system, transcription across targets results in cleavage ofthe target DNA and its transcripts, mediated by independent active siteswithin the Cas10-Csm ribonucleoprotein effector protein complex (see,Samai et al., 2015, Cell, vol. 151, 1164-1174). A CRISPR-Cas system,composition or method targeting RNA via the present effector proteins isthus provided.

In an embodiment, the Cas protein may be a C2c2 ortholog of an organismof a genus which includes but is not limited to Leptotrichia, Listeria,Corynebacter, Sutterella, Legionella, Treponema, Filifactor,Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides,Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma and Campylobacter. Species of organism ofsuch a genus can be as otherwise herein discussed.

Some methods of identifying orthologues of CRISPR-Cas system enzymes mayinvolve identifying tracr sequences in genomes of interest.Identification of tracr sequences may relate to the following steps:Search for the direct repeats or tracr mate sequences in a database toidentify a CRISPR region comprising a CRISPR enzyme. Search forhomologous sequences in the CRISPR region flanking the CRISPR enzyme inboth the sense and antisense directions. Look for transcriptionalterminators and secondary structures. Identify any sequence that is nota direct repeat or a tracr mate sequence but has more than 50% identityto the direct repeat or tracr mate sequence as a potential tracrsequence. Take the potential tracr sequence and analyze fortranscriptional terminator sequences associated therewith.

It will be appreciated that any of the functionalities described hereinmay be engineered into CRISPR enzymes from other orthologs, includingchimeric enzymes comprising fragments from multiple orthologs. Examplesof such orthologs are described elsewhere herein. Thus, chimeric enzymesmay comprise fragments of CRISPR enzyme orthologs of an organism whichincludes but is not limited to Leptotrichia, Listeria, Corynebacter,Sutterella, Legionella, Treponema, Filifactor, Eubacterium,Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola,Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter,Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor,Mycoplasma and Campylobacter. A chimeric enzyme can comprise a firstfragment and a second fragment, and the fragments can be of CRISPRenzyme orthologs of organisms of genera herein mentioned or of speciesherein mentioned; advantageously the fragments are from CRISPR enzymeorthologs of different species.

In embodiments, the CRISPR effector protein as referred to herein alsoencompasses a functional variant of the CRISPR effector or a homologueor an orthologue thereof. A “functional variant” of a protein as usedherein refers to a variant of such protein which retains at leastpartially the activity of that protein. Functional variants may includemutants (which may be insertion, deletion, or replacement mutants),including polymorphs, etc. Also included within functional variants arefusion products of such protein with another, usually unrelated, nucleicacid, protein, polypeptide or peptide. Functional variants may benaturally occurring or may be man-made. Advantageous embodiments caninvolve engineered or non-naturally occurring Type VI RNA-targetingeffector protein.

In an embodiment, nucleic acid molecule(s) encoding the CRISPR effectoror an ortholog or homolog thereof, may be codon-optimized for expressionin a eukaryotic cell. A eukaryote can be as herein discussed. Nucleicacid molecule(s) can be engineered or non-naturally occurring.

In an embodiment, the CRISPR effector or an ortholog or homolog thereof,may comprise one or more mutations (and hence nucleic acid molecule(s)coding for same may have mutation(s). The mutations may be artificiallyintroduced mutations and may include but are not limited to one or moremutations in a catalytic domain. Examples of catalytic domains withreference to a Cas9 enzyme may include but are not limited to RuvC I,RuvC II, RuvC III and HNH domains.

In an embodiment, the CRISPR effector or an ortholog or homolog thereof,may comprise one or more mutations. The mutations may be artificiallyintroduced mutations and may include but are not limited to one or moremutations in a catalytic domain. Examples of catalytic domains withreference to a Cas enzyme may include but are not limited to HEPNdomains.

In an embodiment, the CRISPR effector or an ortholog or homolog thereof,may be used as a generic nucleic acid binding protein with fusion to orbeing operably linked to a functional domain. Exemplary functionaldomains may include but are not limited to translational initiator,translational activator, translational repressor, nucleases, inparticular ribonucleases, a spliceosome, beads, a lightinducible/controllable domain or a chemically inducible/controllabledomain.

In certain example embodiments, the CRISPR effector protein, inparticular the C2c2 protein may be from an organism selected from thegroup consisting of; Leptotrichia, Listeria, Corynebacter, Sutterella,Legionella, Treponema, Filifactor, Eubacterium, Streptococcus,Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium,Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia,Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, andCampylobacter.

In certain embodiments, the effector protein may be a Listeria sp.C2c2p, preferably Listeria seeligeria C2c2p, more preferably Listeriaseeligeria serovar 1/2b str. SLCC3954 C2c2p and the crRNA sequence maybe 44 to 47 nucleotides in length, with a 5′ 29-nt direct repeat (DR)and a 15-nt to 18-nt spacer.

In certain embodiments, the effector protein may be a Leptotrichia sp.C2c2p, preferably Leptotrichia shahii C2c2p, more preferablyLeptotrichia shahii DSM 19757 C2c2p and the crRNA sequence may be 42 to58 nucleotides in length, with a 5′ direct repeat of at least 24 nt,such as a 5′ 24-28-nt direct repeat (DR) and a spacer of at least 14 nt,such as a 14-nt to 28-nt spacer, or a spacer of at least 18 nt, such as19, 20, 21, 22, or more nt, such as 18-28, 19-28, 20-28, 21-28, or 22-28nt.

In certain example embodiments, the effector protein may be aLeptotrichia sp., Leptotrichia wadei F0279, or a Listeria sp.,preferably Listeria newyorkensis FSL M6-0635.

In certain example embodiments, the C2c2 effector proteins of theinvention include, without limitation, the following 21 ortholog species(including multiple CRISPR loci: Leptotrichia shahii; Leptotrichia wadei(Lw2); Listeria seeligeri; Lachnospiraceae bacterium MA2020;Lachnospiraceae bacterium NK4A179; [Clostridium] aminophilum DSM 10710;Carnobacterium gallinarum DSM 4847; Carnobacterium gallinarum DSM 4847(second CRISPR Loci); Paludibacter propionicigenes WB4; Listeriaweihenstephanensis FSL R9-0317; Listeriaceae bacterium FSL M6-0635;Leptotrichia wadei F0279; Rhodobacter capsulatus SB 1003; Rhodobactercapsulatus R121; Rhodobacter capsulatus DE442; Leptotrichia buccalisC-1013-b; Herbinix hemicellulosilytica; [Eubacterium] rectale;Eubacteriaceae bacterium CHKCI004; Blautia sp. Marseille-P2398; andLeptotrichia sp. oral taxon 879 str. F0557. Twelve (12) furthernon-limiting examples are: Lachnospiraceae bacterium NK4A144;Chloroflexus aggregans; Demequina aurantiaca; Thalassospira sp. TSL5-1;Pseudobutyrivibrio sp. OR37; Butyrivibrio sp. YAB3001; Blautia sp.Marseille-P2398; Leptotrichia sp. Marseille-P3007; Bacteroides ihuae;Porphyromonadaceae bacterium KH3CP3RA; Listeria riparia; andInsolitispirillum peregrinum.

In certain embodiments, the C2c2 protein according to the invention isor is derived from one of the orthologues as described in Table 4 below,or is a chimeric protein of two or more of the orthologues as describedin Table 4 below, or is a mutant or variant of one of the orthologues asdescribed in Table 4 below (or a chimeric mutant or variant), includingdead C2c2, split C2c2, destabilized C2c2, etc. as defined hereinelsewhere, with or without fusion with a heterologous/functional domain.

In certain example embodiments, the C2c2 effector protein is selectedfrom Table 4 below.

TABLE 4 C2c2 orthologue Code Multi Letter Leptotrichia shahii C2-2 LshL. wadei F0279 (Lw2) C2-3 Lw2 Listeria seeligeri C2-4 LseLachnospiraceae bacterium MA2020 C2-5 LbM Lachnospiraceae bacteriumNK4A179 C2-6 LbNK179 Clostridium aminophilum DSM 10710 C2-7 CaCarnobacterium gallinarum DSM 4847 C2-8 Cg Carnobacterium gallinarum DSM4847 C2-9 Cg2 Paludibacter propionicigenes WB4 C2-10 Pp Listeriaweihenstephanensis FSL R9-0317 C2-11 Lwei Listeriaceae bacterium FSLM6-0635 C2-12 LbFSL Leptotrichia wadei F0279 C2-13 Lw Rhodobactercapsulatus SB 1003 C2-14 Re Rhodobacter capsulatus R121 C2-15 ReRhodobacter capsulatus DE442 C2-16 Re Leptotrichia buccalis C-1013-bC2-17 LbuC2c2 Herbinix hemicellulosilytics C2-18 HheC2c2 Eubacteriumrectale C2-19 EreC2c2 Eubacteriaceae bacterium CHKC1004 C2-20 EbaC2c2Blautia sp. Marseille-P2398 C2-21 BsmC2c2 Leptotrichia sp. oral taxon879 str. F0557 C2-22 LspC2c2 Lachnospiraceae bacterium NK4a144Chloroflexus aggregans Demequina aurantiaca Thalassospira sp. TSL5-1Pseudobutyrivibrio sp. 0R37 Butyrivibrio sp. YAB3001 Blautia sp.Marseille-P2398 Leptotrichia sp. Marseille-P300 Bacteroides ihuaePorphyromonadaceae bacterium KH3CP3RA Listeria riparia Insolitispirillumperegrinum

The wild type protein sequences of the above species are listed in Table5 below. In certain embodiments, a nucleic acid sequence encoding theC2c2 protein is provided.

TABLE 5 C2c2-2 L. shahiimgnlfghkrwyevrdkkdfkikrkvkvkrnydgnkyilninennnkekidnnkfirkyi (Lsh)nykkndnilkeftrkfhagnilfklkgkegiiriennddfleteevvlyieaygkseklkalgi(SEQ. I.D.tkkkiideairqgitkddkkieiknieneeeieidirdeytnktlndcsiilriiendeletkksiNo. 1) yeifkninmslykiiekiienetekvfenryyeehlrekllkddkidviltnfmeirekiksnleilgfvkfylnvggdkkksknkkmlvekilninvdltvediadfvikelefwnitkriekvkkvnneflekrrnrtyiksyylldkhekfkierenkkdkivkffveniknnsikekiekilaefkidelikklekelkkgncdteifgifkkhykvnfdskkfskksdeekelykiiyrylkgriekilvneqkvrlkkmekieiekilnesilsekilkrvkqytlehimylgklrhndidmttvntddfsrlhakeeldlelitffastnmelnkifsreninndenidffggdreknyvldkkilnskikiirdldfidnknnitnnfirkftkigtnernrilhaiskerdlqgtqddynkviniignikisdeevskalnldvvflcdkkniitkindikiseennndikylpsfskylpeilnlyrnnpknepfdtietekivlnaliyvnkelykklileddleeneskniflgelkktlgnideideniienyyknaqisaskgnnkaikkyqkkviecyigylrknyeelfdfsdfkmniqeikkqikdindnktyeritvktsdktivinddfeyiisifallnsnavinkirnrffatsvwlntseyqniidildeimqlntlrnecitenwnlnleefiqkmkeiekdfddfkiqtkkeifnnyyediknniltefkddingcdvlekklekivifddetkfeidkksnilqdeqrklsninkkdlkkkvdqyikdkdqeikskilcriifnsdflkkykkeidnliedmesenenkfqeiyypkerknelyiykknlflnignpnfdkiyglisndikmadakflfnidgknirknkiseidailknlndklngyskeykekyikklkenddffakniqnknyksfekdynryseykkirdlvefnylnkiesylidinwklaiqmarferdmhyivnglrelgiiklsgyntgisraypkrngsdgfytttayykffdeesykkfekicygfgidlsenseinkpenesirnyishfyivrnpfadysiaeqidrvsnllsystrynnstyasvfevfkkdvnldydelkkkfklignndilerlmkpkkvsvlelesynsdyiknliielltkientndtlkrpaatkkagqakkkkgsypydvpdyaypydvpdyaypydvpdya c2c2-3 L. wadeimkvtkvdgishkkyieegklykstseenrtserlsellsirldiyiknpdnaseeenrirrenl (Lw2)kkffsnkvlhlkdsvlylknrkeknavqdknyseediseydlknknsfsvlkkillnedvn (SEQ. I.D.seeleifrkdveaklnkinslkysfeenkanyqkinennvekvggkskrniiydyyresak No. 2)rndyinnvqeafdklykkedieklfflienskkhekykireyyhkiigrkndkenfakiiyeeiqnvnnikeliekipdmselkksqvfykyyldkeelndknikyafchfveiemsqllknyvykrlsnisndkikrifeyqnlkklienkllnkldtyvrncgkynyylqvgeiatsdfiarnrqneaflrniigvssvayfslrniletenenditgrmrgktvknnkgeekyvsgevdkiynenkqnevkenlkmfysydfnmdnkneiedffanideaissirhgivhfnlelegkdifafkniapseiskkmfqneinekklklkifkqlnsanvfnyyekdviikylkntkfnfvnknipfvpsftklynkiedlrntlkffwsvpkdkeekdaqiyllkniyygeflnkfvknskvfflcitnevikinkqrnqktghykyqkfeniektvpveylaiiqsreminnqdkeekntyidfiqqiflkgfidylnknnlkyiesnnnndnndifskikikkdnkekydkilknyekhnrnkeipheinefvreiklgkilkytenlnmfylilkllnhkeltnlkgslekyqsankeetfsdelelinllnldnnrytedfeleaneigkfldfnenkikdrkelkkfdtnkiyfdgeniikhrafynikkygmlnllekiadkakykislkelkeysnkkneieknytmqqnlhrkyarpkkdekfndedykeyekaigniqkythlknkvefnelnllqglllkilhrlvgytsiwerdlrfrlkgefpenhyieeifnfdnsknvkyksgqivekyinfykelykdnvekrsiysdkkvkklkqekkdlyirnyiahfnyiphaeisllevlenlrkllsydrklknaimksivdilkeygfvatflcigadkkieiqtlesekivhlknlkkkklmtdrnseelcelvkvmfeykalekrpaatkkagqakkkkgsypydvpdyaypydvpdyaypydvpdya* c2c2-4 Listeriamwisiktlihhlgvlffedymynrreldciievktmritkvevdrkkvlisrdknggklvye seeligerinemqdnteqimhhkkssfyksvvnkticrpeqkqmkklvhgllqensqekikvsdvtk (SEQ. I.D.lnisnflnhrfkkslyyfpenspdkseeyrieinlsqlledslkkqqgtficwesfskdmely No. 3)inwaenyissktklikksirnnriqstesrsgqlmdrymkdilnknkpfdiqsvsekyqlekltsalkatfkeakkndkeinyklkstlqnherqiieelkenselnqfnieirkhletyfpikktnrkvgdirnleigeiqkivnhrlknkivqrilqegklasyeiestvnsnslqkikieeafalkfinaclfasnnlrnmvypvckkdilmigefknsfkeikhkkfirqwsqffsqeitvddielaswglrgaiapirneiihlkkhswkkffnnptfkvkkskiingktkdvtseflyketlflcdyfyseldsvpeliinkmesskildyyssdqlnqvftipnfelslltsavpfapsflcrvylkgfdyqnqdeaqpdynlklniynekafnseafqaqyslfkmvyyqvflpqfttnndlfkssvdfiltlnkerkgyakafqdirkmnkdekpseymsyiqsqlmlyqkkqeekekinhfekfinqvfikgfnsfieknatyichptkntvpendnieipfhtdmddsniafwlmcklldakqlselrnemikfscslqsteeistftkareviglallngekgcndwkelfddkeawkknmslyvseellqslpytqedgqtpvinrsidlykkygtetileklfsssddykvsakdiaklheydvtekiaqqeslhkqwiekpglardsawtkkyqnvindisnyqwaktkveltqvrhlhqltidllsrlagymsiadrdfqfssnyilerenseyrvtswillsenknknkyndyelynlknasikvsskndpqlkvdlkqlrltleylelfdnrlkekrnnishfnylngqlgnsilelfddardvlsydrklknayskslkeilsshgmevtflcplyqtnhhlkidklqpkkihhlgekstvssnqvsneycql vrtlltmkc2c2-5  1 Lachno-mqiskvnhkhvavgqkdreritgfiyndpvgdeksledvvakrandtkvlfnvfntkdly spiraceaedsqesdksekdkeiiskgakfvaksfnsaitilkkqnkiystltsqqvikelkdkfggariydbacteriumddieealtetlkksfrkenvrnsikvlienaagirsslskdeeeliqeyfvkqlveeytktklq MA2020knvyksiknqnmviqpdsdsqvlslsesrrekqssayssdtlynckekdvlkafltdyavl (SEQ. I.D.dedernsllwklrnlvnlyfygsesirdysytkeksvwkehdeqkanktlfideichitkig No. 4)kngkeqkvldyeenrsrcrkqninyyrsalnyaknntsgifenedsnhfwihlieneverlyngiengeefkfetgyisekvwkavinhlsikyialgkavynyamkelsspgdiepgkiddsyingitsfdyeiikaeeslqrdismnvvfatnylacatvdtdkdfllfskedirsctkkdgnlcknimqfwggystwknfceeylkddkdalellyslksmlysmrnssfhfstenvdngswdteligklfeedcnraariekekfynnnlhmfysssllekvlerlysshherasqvpsfnrvfvrknfpsslseqritpkftdskdeqiwqsavyylckeiyyndflqskeayklfregvknldkndinnqkaadsflcqavvyygkaignatlsqvcqaimteynrqnndglkkksayaekqnsnkykhyplflkqvlqsafweyldenkeiygfisaqihksnveikaedfianyssqqykklvdkvkktpelqkwytlgrlinprqanqflgsirnyvqfvkdiqrrakengnpirnyyevlesdsiikilemakingttsndihdyfrdedeyaeyisqfvnfgdvhsgaalnafcnsesegkkngiyydginpivnrnwvlcklygspdliskiisrvnenmihdfhkqedlireyqikgicsnkkeqqdlrtfqvlknrvelrdiveyseiinelygglikwcylrerdlmyfqlgfhylclnnasskeadyikinvddrnisgailyqiaamyinglpvyykkddmyvalksgkkasdelnsneqtskkinyflkygnnilgdkkdqlylaglelfenvaeheniiifrneidhfhyfydrdrsmldlysevfdrfftydmklrknvvnmlynilldhnivssfvfetgekkvgrgdsevikpsakirlranngvssdvftykvgskdelkiatlpakneefllnvarliyypdmeavsenmvregvvkveksndkkgkisrgsntrssnqskynnksknrmnysmgsifekmdlkfd c2c2-6  2 Lachno-mkiskvreenrgakltvnaktavvsenrsqegilyndpsrygksrkndedrdryiesrlks spiraceaesgklyrifnedknkretdelqwflseivkkinrrnglvlsdmlsvddrafekafekyaelsytbacteriumnrrnkvsgspafetcgvdaataerlkgiisetnfinriknnidnkvsediidriiakylkkslcrNK4A179 ervkrglkkllmnafdlpysdpdidvqrdfidyvledfyhvraksqvsrsiknmnmpvq(SEQ. I.D. pegdgkfaitvskggtesgnkrsaekeafkkflsdyasldervrddmlrrmrrlvvlyfygNo. 5) sddsklsdvnekfdvwedhaarrvdnrefiklplenklangktdkdaerirkntykelyrnqnigcyrqavkaveednngryfddkmlnmffihrieygvekiyanlkqvteflcartgylsekiwkdlinyisikyiamgkavynyamdelnasdkkeielgkiseeylsgissfdyelikaeemlqretavyvafaarhlssqtveldsensdflllkpkgtmdkndknklasnnilnflkdketlrdtilqyfgghslwtdfpfdkylaggkddvdfltdlkdviysmrndsfhyatenhnngkwnkelisamfehetermtvvmkdkfysnnlpmfyknddlkkllidlykdnverasqvpsfnkvfvrknfpalvrdkdnlgieldlkadadkgenelkfynalyymflceiyynaflndknvrerfitkatkvadnydrnkernlkdriksagsdekkklreqlqnyiaendfgqriknivqvnpdytlaqicqlimteynqqnngcmqkksaarkdinkdsyqhykmlllvnlrkaflefikenyafvlkpykhdlcdkadfvpdfakyvkpyaglisrvagsselqkwyivsrflspaqanhmlgflhsykqyvwdiyrrasetgteinhsiaedkiagvditdvdavidlsvklcgtisseisdyflcddevyaeyissyldfeydggnykdslnrfcnsdavndqkvalyydgehpklnrniilsklygerrflekitdrvsrsdiveyyklkketsqyqtkgifdsedeqknikkfqemknivefrdlmdyseiadelqgqlinwiylrerdlmnfqlgyhyaclnndsnkqatyvtldyqgkknrkingailyqicamyinglplyyvdkdssewtvsdgkestgakigefyryaksfentsdcyasgleifenisehdnitelrnyiehfryyssfdrsflgiysevfdrfftydlkyrknvptilynillqhfvnvrfefvsgkkmigidkkdrkiakekecaritirekngvyseqftyklkngtvyvdardkrylqsiirllfypekvnmdemievkekkkpsdnntgkgyskrdrqqdrkeydkykekkkkegnflsgmggninwdeinaqlkn c2c2-7  3 Clostridiummkfskvdhtrsavgiqkatdsvhgmlytdpkkqevndldkrfdqlnvkakrlynvfnqs aminophilumkaeedddekrfgkvvkklnrelkdllfhrevsrynsignakynyygiksnpeeivsnlgm DSMveslkgerdpqkvisklllyylrkglkpgtdglrmileascglrklsgdekelkvflqtldedf 10710ekktfkknlirsienqnmavqpsnegdpiigitqgrfnsqkneeksaiermmsmyadln (SEQ. I.D.edhredvlrklrrinvlyfnvdtekteeptlpgevdtnpvfevwhdhekgkendrqfatfa No. 6)kiltedretrkkeklavkealndlksairdhnimayrcsikvteqdkdglffedqrinrfwihhiesaverilasinpeklyklrigylgekvwkdllnylsikyiavgkavfhfamedlgktgqdielgklsnsysggltsfdyeqiradetlqrqlsvevafaannlfravvgqtgkkieqskseeneedfllwkaekiaesikkegegntlksilqffggasswdlnhfcaaygnessalgyetkfaddlrkaiyslrnetfhfttlnkgsfdwnakligdmfsheaatgiavertrfysnnlpmfyresdlkrimdhlyntyhprasqvpsfnsvfvrknfrlflsntlntntsfdtevyqkwesgvyylfkeiyynsflpsgdahhlffeglrrirkeadnlpivgkeakkrnavqdfgrrcdelknlslsaicqmimteyneqnngnrkykstredkrkpdifqhykmlllrtlqeafaiyirreeflcfifdlpktlyvmkpveeflpnwksgmfdslvervkqspdlqrwyvlckflngrllnqlsgvirsyiqfagdiqrrakanhnrlymdntqrveyysnvlevvdfcikgtsrfsnvfsdyfrdedayadyldnylqflcdekiaevssfaalktfcneeevkagiymdgenpvmqrnivmaklfgpdevlknvvpkvtreeieeyyqlekqiapyrqngyckseedqkkllrfqriknrvefqtitefseiinellgqliswsflrerdllyfqlgfhylclhndtekpaeykeisredgtvirnailhqvaamyvgglpvytladkklaafekgeadcklsiskdtagagkkikdffryskyvlikdrmltdqnqkytiylaglelfentdehdnitdvrkyvdhfkyyatsdenamsildlyseihdrfftydmkyqknvanmlenillrhfvlirpefftgskkvgegkkitckaraqieiaengmrsedftyklsdgkknistcmiaardqkylntvarllyypheakksivdtrekknnkktnrgdgtfnkqkgtarkekdngprefndtgfsntpfagfdpfrns c2c2-8  5 Carnomritkvkikldnklyqvtmqkeekygtlklneesrkstaeilrlkkasfnksfhsktinsqk bacteriumenknatikkngdyisqifeklvgvdtnknirkpkmsltdlkdlpkkdlalfikrkfknddivgallinarum eiknldlislfynalqkvpgehftdeswadfcqemmpyreyknkfierkiillansieqnkDSM 4847 gfsinpetfskrkrvlhqwaievqergdfsildeklsklaeiynfkkmckrvqdelndleks(SEQ. I.D. mkkgknpekekeaykkqknflciktiwkdypykthigliekikeneelnqfnieigkyfeNo. 7) hyfpikkerctedepyylnsetiattvnyqlknalisylmqigkykqfglenqvldskklqeigiyegfqtkfmdacvfatsslkniiepmrsgdilgkrefkeaiatssfvnyhhffpyfpfelkgmkdreselipfgeqteakqmqniwalrgsvqqirneifhsfdknqkfnlpqldksnfefdasenstgksqsyietdykflfeaeknqleqffierikssgaleyyplksleklfakkemkfslgsqvvafapsykklvkkghsyqtategtanylglsyynryelkeesfqaqyyllkliyqyvflpnfsqgnspafretvkailrinkdearkkmkknkkflrkyafeqvremefketpdqymsylqsemreekvrkaekndkgfeknitmnfekllmqifvkgfdvflttfagkelllsseekviketeislskkinerektlkasiqvehqlvatnsaisywlfcklldsrhlnelrnemikflcqsrikfnhtqhaeliqnllpiveltilsndydekndsqnvdvsayfedkslyetapyvqtddrtrvsfrpilklekyhtkslieallkdnpqfrvaatdiqewmhkreeigelvekrknlhtewaegqqtlgaekreeyrdyckkidrfnwkankvtltylsqlhylitdllgrmvgfsalferdlvyfsrsfselggetyhisdyknlsgvlrinaevkpikiknikvidneenpykgnepevkpfldrlhaylenvigikavhgkirnqtahlsvlqlelsmiesmnnlrdlmaydrklknavtksmikildkhgmilklkidenhknfeieslipkeiihlkdkaiktnqvseeycqlvlallttnpgnqln c2c2-9 6 Carno- mrmtkvkingspvsmnrsklnghlywngttntvniltkkeqsfaasflnktivkadqvkgbacteriumykvlaenifiifeqleksnsekpsvylnnirrlkeaglkrfflcskyheeikytseknqsvptklgallinarum nliplffnavdriqedkfdeknwsyfckemspyldykksylnrkkeilansiqqnrgfsmDSM 4847 ptaeepnllskrkqlfqqwamkfqespliqqnnfaveqfnkefankinelaavynvdelc(SEQ. I.D. taiteklmnfdkdksnktrnfeikklwkqhphnkdkaliklfnqegnealnqfnielgkyfNo. 8) ehyfpktgkkesaesyylnpqtiiktvgyqlrnafvqyllqvgklhqynkgvldsqtlqeigmyegfqtkfmdacvfassslrniiqattnediltrekflckeleknvelkhdlffkteiveerdenpakkiamtpneldlwairgavqrvrnqifhqqinkrhepnqlkvgsfengdlgnvsyqktiyqklfdaeikdieiyfaekikssgaleqysmkdleklfsnkeltlslggqvvafapsykklykqgyfyqnektieleqftdydfsndvfkanyylikliyhyvflpqfsgannklfkdtvhyviqqnkelnttekdkknnkkirkyafeqvklmknespekymqylqremqeertikeakktneekpnynfeklliqifikgfdtflrnfdlnlnpaeelvgtvkekaeglrkrkeriakilnvdeqiktgdeeiafwifaklldarhlselrnemikflcqssvkkglikngdlieqmqpilelcilsndsesmekesfdkievflekvelaknepymqedkltpvkfrfmkqlekyqtrnfienlvienpefkvsekivinwheekekiadlvdkrtklheewaskareieeynekikknkskkldkpaefakfaeykiiceaienfnrldhkvrltylknlhylmidlmgrmvgfsvlferdfvymgrsysalkkqsiylndydtfanirdwevnenkhlfgtsssdltfqetaefknlkkpmenqlkallgvtnhsfeirnniahlhvlrndgkgegvsllscmndlrklmsydrklknavtkaiikildkhgmilkltnndhtkpfeieslkpkkiihleksnhsfpmdqvsqeycdlvkkmlvftn c2c2-10  7Paludibactermrvskykykdggkdkmvlvhrkttgaqlvysgqpvsnetsnilpekkrqsfdlstlnktiipropionicigeneskfdtakkqklnvdqykivekificypkqelpkqikaeeilpflnhkfqepvkywkngkee WB4sfnitlliveavqaqdkrklqpyydwktwyiqtksdllkksiennridltenlskrkkallaw(SEQ. I.D. eteftasgsidlthyhkvymtdvlckmlqdvkpltddkgkintnayhrglkkalqnhqpaNo. 9) ifgtrevpneanradnqlsiyhlevvkylehyfpiktskrrntaddiahylkaqtlkttiekqlvnairaniiqqgktnhhelkadttsndliriktneafvinitgtcafaannirnmvdneqtndilgkgdfiksllkdntnsqlysfffgeglstnkaeketqlwgirgavqqirnnvnhykkdalktvfnisnfenptitdpkqqtnyadtiykarfinelekipeafaqqlktggaysyytienlksllttfqfslcrstipfapgfkkvfngginyqnakqdesfyelmleqylrkenfaeesynaryfmlkliynnlflpgfttdrkafadsvgfvqmqnkkqaekvnprkkeayafeavrpmtaadsiadymayvqselmqeqnkkeekvaeetrinfekfvlqvfikgfdsflrakefdfvqmpqpqltatasnqqkadklnqleasitadckltpqyakaddathiafyvfcklldaahlsnlrnelikfresvnefkfhhlleiieicllsadvvptdyrdlysseadclarlrpfieqgaditnwsdlfvqsdkhspvihanielsvkygttklleqiinkdtqfktteanftawntaqksieqlikqredhheqwvkaknaddkekqerkreksnfaqkfiekhgddyldicdyintynwldnkmhfvhlnrlhgltiellgrmagfvalfdrdfqffdeqqiadefklhgfvnlhsidkklnevptkkikeiydirnkiiqingnkinesvranliqfisskrnyynnaflhvsndeikekqmydirnhiahfnyltkdaadfslidlinelrellhydrklknayskafidlfdkhgmilklklnadhklkveslepkkiyhlgssakdkpeyqyctnqvmmaycnmcrsllemkk c2c2-11  9 Listeriamlallhqevpsqklhnlkslntesltklfkpkfqnmisyppskgaehvqfcltdiavpaird weihen-ldeikpdwgiffeklkpytdwaesyihykqttiqksieqnkiqspdsprklvlqkyvtaflnstephanensisgeplgldlvakkykladlaesfkvvdlnedksanykikaclqqhqrnildelkedpelnqyFSL R9-0317gievkkyiqryfpikrapnrskharadflkkeliestveqqfknavyhyvleqgkmeayel (SEQ. I.D.tdpktkdlqdirsgeafsfkfinacafasnnlkmilnpecekdilgkgdfkknlpnsttqsd No. 10)vvkkmipffsdeiqnvnfdeaiwairgsiqqirnevyhckkhswksilkikgfefepnnmkytdsdmqklmdkdiakipdfieeklkssgiirfyshdklqsiwemkqgfsllttnapfvpsfkrvyakghdyqtsknryydlglttfdileygeedfraryfltklvyyqqfmpwftadnnafrdaanfvlrinknrqqdakafinireveegemprdymgyvqgqiaihedstedtpnhfekfisqvfikgfdshmrsadlkfiknprnqgleqseieemsfdikvepsflknkddyiafwtfckmldarhlselrnemikydghltgeqeiiglallgvdsrendwkqffssereyekimkgyvgeelyqrepyrqsdgktpilfrgvegarkygtetviqrlfdaspefkvskcnitewerqketieetierrkelhneweknpkkpqnnaffkeykeccdaidaynwhknkttivyvnelhhllieilgryvgyvaiadrdfqcmanqyfkhsgiterveywgdnrlksikkldtflkkeglfvseknarnhiahlnylslksectllylserlreifkydrklknayskslidildrhgmsvvfanlkenkhrlvikslepkklrhlgekkidngyietnqvseeycgivkrllei c2c2-12 10Listeriaceaemkitkmrvdgrtivmertskegqlgyegidgnktteiifdkkkesfyksilnktvrkpdek bacteriumeknrrkqainkainkeitelmlavlhqevpsqklhnlkslntesltklfkpkfqnmisypps FSL M6-kgaehvqfcltdiavpairdldeikpdwgiffeklkpytdwaesyihykqttiqksieqnki 0635 =qspdsprklvlqkyvtaflngeplgldlvakkykladlaesfklvdlnedksanykikaclq Listeriaqhqrnildelkedpelnqygievkkyiqryfpikrapnrskharadflkkeliestveqqfknewyorkensisnavyhyvleqgkmeayeltdpktkdlqdirsgeafsfkfinacafasnnlkmilnpecek FSL M6-dilgkgnfkknlpnsttrsdvvkkmipffsdelqnvnfdeaiwairgsiqqirnevyhckk 0635hswksilkikgfefepnnmkyadsdmqklmdkdiakipefieeklkssgvvrfyrhdel (SEQ. I.D.qsiwemkqgfsllttnapfvpsfkrvyakghdyqtsknryynldlttfdileygeedfrary No. 11)fltklvyyqqfmpwftadnnafrdaanfvlrinknrqqdakafinireveegemprdymgyvqgqiaihedsiedtpnhfekfisqvfikgfdrhmrsanlkfiknprnqgleqseieemsfdikvepsflknkddyiafwifckmldarhlselrnemikydghltgeqeliglallgvdsrendwkqffssereyekimkgyvveelyqrepyrqsdgktpilfrgveqarkygtetviqrlfdanpefkvskcnlaewerqketieetikrrkelhnewaknpkkpqnnaffkeykeccdaidaynwhknkttlayvnelhhllieilgryvgyvaiadrdfqcmanqyfkhsgiterveywgdnrlksikkldtflkkeglfvseknarnhiahlnylslksectllylserlreifkydrklknayskslidildrhgmsvvfanlkenkhrlvikslepkklrhlggkkidggyietnqvseeyc givkrllemc2c2-13 12 Leptotrichiamkvtkvdgishkkyieegklykstseenrtserlsellsirldiyiknpdnaseeenrirrenl wadeikkffsnkvlhlkdsvlylknrkeknavqdknyseediseydlknknsfsvlkkillnedvn F0279seeleifrkdveaklnkinslkysfeenkanyqkinennvekvggkskrniiydyyresak (SEQ. I.D.rndyinnvqeafdklykkedieklfflienskkhekykireyyhkiigrkndkenfakiiye No. 12)eiqnvnnikeliekipdmselkksqvfykyyldkeelndknikyafchfveiemsqllknyvykrlsnisndkikrifeyqnlkklienkllnkldtyvrncgkynyylqvgeiatsdfiarnrqneaflrniigvssvayfslrniletenenditgrmrgktvknnkgeekyvsgevdkiynenkqnevkenlkmfysydfnmdnkneiedffanideaissirhgivhfnlelegkdifafkniapseiskkmfqneinekklklkifkqlnsanvfnyyekdviikylkntkfnfvnknipfvpsftklynkiedlrntlkffwsvpkdkeekdaqiyllkniyygeflnkfvknskvfflcitnevikinkqrnqktghykyqkfeniektvpveylaiiqsreminnqdkeekntyidfiqqiflkgfidylnknnlkyiesnnnndnndifskikikkdnkekydkilknyekhnrnkeipheinefvreiklgkilkytenlnmfylilkllnhkeltnlkgslekyqsankeetfsdelelinllnldnnrytedfeleaneigkfldfnenkikdrkelkkfdtnkiyfdgeniikhrafynikkygmlnllekiadkakykislkelkeysnkkneieknytmqqnlhrkyarpkkdekfndedykeyekaigniqkythlknkvefnelnllqglllkilhrlvgytsiwerdlrfrlkgefpenhyieeifnfdnsknvkyksgqivekyinfykelykdnvekrsiysdkkvkklkqekkdlyirnyiahfnyiphaeisllevlenlrkllsydrklknaimksivdilkeygfvatflcigadkkieiqtlesekivhlknlkkkklmtdrnseelcelvkvmfeykale c2c2-14 15 Rhodobactermqigkvqgrtisefgdpagglkrkistdgknrkelpahlssdpkaligqwisgidkiyrkp capsulatusdsrksdgkaihsptpskmqfdarddlgeafwklvseaglaqdsdydqfkrrlhpygdkf SB 1003qpadsgaklkfeadppepqafhgrwygamskrgndakelaaalyehlhvdekridgqp (SEQ. I.D.krnpktdkfapglvvaralgiessylprgmarlarnwgeeeiqtyfvvdvaasykevaka No. 13)aysaaqafdpprqvsgrslspkvgfalaehlervtgskrcsfdpaagpsvlalhdevkktykrlcargknaarafpadktellalmrhthenrvrnqmvrmgryseyrgqqagdlaqshywtsagqteikeseifvrlwvgafalagrsmkawidpmgkivntekndrdltaavnirqvisnkemvaeamarrgiyfgetpeldrlgaegnegfvfallrylrgcrnqtfhlgaragflkeirkelektrwgkakeaehvyltdktvaairaiidndakalgarlladlsgafvahyaskehfstlyseivkavkdapevssglprlklllkradgvrgyvhglrdtrkhafatklppppaprelddpatkaryiallrlydgpfrayasgitgtalagpaarakeaatalaqsvnvtkaysdvmegrtsrlrppndgetlreylsaltgetatefrvqigyesdsenarkqaefienyrrdmlafmfedyirakgfdwilkiepgatamtrapvlpepidtrgqyehwqaalylvmhfvpasdvsnllhqlrkwealqgkyelvqdgdatdqadarrealdlykrfrdvlvlflktgearfegraapfdlkpfralfanpatfdrlfmatpttarpaeddpegdgasepelrvartlrglrqiarynhmavlsdlfakhkvrdeevarlaeiedetqeksqivaagelrtdlhdkvmkchpktispeerqsyaaaiktieehrflvgrvylgdhlrlhrlmmdvigrlidyagayerdtgtflinaskqlgagadwavtiagaantdartqtrkdlahfnvldradgtpdltalvnraremmaydrkrknavprsildmlarlgltlkwqmkdhllqdatitqaaikhldkvrltvggpaavtearfsqdylqmvaavfngsvqnpkprrrddgdawhkppkpataqsqpdqkppnkapsagsrlpppqvgevyegvvvkvidtgslgflavegvagniglhisrlrriredaiivgrryrfrveiyvppksntsklnaadlvrid c2c2-1516 Rhodobactermqigkvqgrtisefgdpagglkrkistdgknrkelpahlssdpkaligqwisgidkiyrkp capsulatusdsrksdgkaihsptpskmqfdarddlgeafwklvseaglaqdsdydqfkrrlhpygdkf R121 (SEQ.qpadsgaklkfeadppepqafhgrwygamskrgndakelaaalyehlhvdekridgqp I.D. No. 14)krnpktdkfapglvvaralgiessylprgmarlarnwgeeeiqtyfvvdvaasykevakaaysaaqafdpprqvsgrslspkvgfalaehlervtgskrcsfdpaagpsvlalhdevkktykrlcargknaarafpadktellalmrhthenrvrnqmvrmgrvseyrgqqagdlaqshywtsagqteikeseifvrlwvgafalagrsmkawidpmgkivntekndrdltaavnirqvisnkemvaeamarrgiyfgetpeldrlgaegnegfvfallrylrgcrnqtfhlgaragflkeirkelektrwgkakeaehvvltdktvaairaiidndakalgarlladlsgafvahyaskehfstlyseivkavkdapevssglprlklllkradgvrgyvhglrdtrkhafatklppppaprelddpatkaryiallrlydgpfrayasgitgtalagpaarakeaatalaqsvnvtkaysdvmegrssrlrppndgetlreylsaltgetatefrvqigyesdsenarkqaefienyrrdmlafmfedyirakgfdwilkiepgatamtrapvlpepidtrgqyehwqaalylvmhfvpasdvsnllhqlrkwealqgkyelvqdgdatdqadarrealdlykrfrdvlvlflktgearfegraapfdlkpfralfanpatfdrlfmatpttarpaeddpegdgasepelrvartlrglrqiarynhmavlsdlfakhkvrdeevarlaeiedetqeksqivaagelrtdlhdkvmkchpktispeerqsyaaaiktieehrflvgrvylgdhlrlhrlmmdvigrlidyagayerdtgtflinaskqlgagadwavtiagaantdartqtrkdlahfnvldradgtpdltalvnraremmaydrkrknavprsildmlarlgltlkwqmkdhllqdatitqaaikhldkvrltvggpaavtearfsqdylqmvaavfngsvqnpkprrrddgdawhkppkpataqsqpdqkppnkapsagsrlpppqvgevyegvvvkvidtgslgflavegvagniglhisrlrriredaiivgrryrfrveiyvppksntsklnaadlvrid c2c2-1617 Rhodobactermqigkvqgrtisefgdpagglkrkistdgknrkelpahlssdpkaligqwisgidkiyrkp capsulatusdsrksdgkaihsptpskmqfdarddlgeafwklvseaglaqdsdydqfkrrlhpygdkf DE442qpadsgaklkfeadppepqafhgrwygamskrgndakelaaalyehlhvdekridgqp (SEQ. ID.krnpktdkfapglvvaralgiessvlprgmarlarnwgeeeiqtyfvvdvaasvkevaka No. 15)aysaaqafdpprqvsgrslspkvgfalaehlervtgskrcsfdpaagpsvlalhdevkktykrlcargknaarafpadktellalmrhthenrvrnqmvrmgrvseyrgqqagdlaqshywtsagqteikeseifvrlwvgafalagrsmkawidpmgkivntekndrdltaavnirqvisnkemvaeamarrgiyfgetpeldrlgaegnegfvfallrylrgcrnqtfhlgaragflkeirkelektrwgkakeaehvvltdktvaairaiidndakalgarlladlsgafvahyaskehfstlyseivkavkdapevssglprlklllkradgvrgyvhglrdtrkhafatklppppaprelddpatkaryiallrlydgpfrayasgitgtalagpaarakeaatalaqsvnvtkaysdvmegrssrlrppndgetlreylsaltgetatefrvqigyesdsenarkqaefienyrrdmlafmfedyirakgfdwilkiepgatamtrapvlpepidtrgqyehwqaalylvmhfvpasdvsnllhqlrkwealqgkyelvqdgdatdqadarrealdlykrfrdvlvlflktgearfegraapfdlkpfralfanpatfdrlfmatpttarpaeddpegdgasepelrvartlrglrqiarynhmavlsdlfakhkvrdeevarlaeiedetqeksqivaagelrtdlhdkvmkchpktispeerqsyaaaiktieehrflvgrvylgdhlrlhrlmmdvigrlidyagayerdtgtflinaskqlgagadwavtiagaantdartqtrkdlahfnvldradgtpdltalvnraremmaydrkrknavprsildmlarlgltlkwqmkdhllqdatitqaaikhldkvrltvggpaavtearfsqdylqmvaavfngsvqnpkprrrddgdawhkppkpataqsqpdqkppnkapsagsrlpppqvgevyegvvvkvidtgslgflavegvagniglhisrlrriredaiivgrryrfrveiyvppksntsklnaadlvrid LbuC2c2Leptorichiamkvtkvggishkkytsegrlvkseseenrtderlsallnmrldmyiknpsstetkenqkribuccalis C-gklkkffsnkmvylkdntlslkngkkenidreysetdilesdvrdkknfavlkkiylnenv 1013-bnseelevfrndikkklnkinslkysfeknkanyqkinenniekvegkskrniiydyyresa (SEQ IDkrdayvsnvkeafdklykeediaklvleienitklekykirefyheiigrkndkenfakiiye NO: 16)eiqnvnnmkeliekvpdmselkksqvfykyyldkeelndknikyafchfveiemsqllknyvykrlsnisndkikrifeyqnlkklienkllnkldtyvrncgkynyylqdgeiatsdfiarnrqneaflrniigvssvayfslrniletenenditgrmrgktvknnkgeekyvsgevdkiynenkknevkenlkmfysydfnmdnkneiedffanideaissirhgivhfnlelegkdifafkniapseiskkmfqneinekklklkifrqlnsanvfrylekykilnylkrtrfefvnknipfvpsftklysriddlknslgiywktpktnddnktkeiidaqiyllkniyygeflnyfmsnngnffeiskeiielnkndkrnlktgfyklqkfediqekipkeylaniqslyminagnqdeeekdtyidfiqkiflkgfmtylanngrlsliyigsdeetntslaekkqefdkflkkyeqnnnikipyeineflreiklgnilkyterinmfylilkllnhkeltnlkgslekyqsankeeafsdqlelinllnldnnrytedfeleadeigkfldfngnkvkdnkelkkfdtnkiyfdgeniikhrafynikkygmlnllekiadkagykisieelkkysnkkneieknhkmqenlhrkyarprkdekftdedyesykqaienieeythlknkvefnelnllqglllrilhrlvgytsiwerdlrfrlkgefpenqyieeifnfenkknvkykggqivekyikfykelhqndevkinkyssanikvlkqekkdlyirnyiahfnyiphaeisllevlenlrkllsydrklknavmksvvdilkeygfvatflcigadkkigiqtlesekivhlknlkkkklmtdrnseelcklvkimfeykmeekksen HheC2c2 Herbinixmkltrrrisgnsvdqkitaafyrdmsqgllyydsedndctdkviesmdferswrgrilknghemicelluloseddknpfymfvkglvgsndkivcepidvdsdpdnldilinknitgfgrnlkapdsndtleilytica (SEQnlirkiqagipeeevlpelkkikemiqkdivnrkeqllksiknnripfslegsklvpstkkmID NO: 17) kwlfklidvpnktfnekmlekyweiydydklkanitnrldktdkkarsisravseelreyhknlrtnynrfvsgdrpaagldnggsakynpdkeefllflkeveqyfkkyfpvkskhsnkskdkslvdkyknycsykvvkkevnrsiinqlvagliqqgkllyyfyyndtwqedflnsyglsyiqveeafkksvmtslswginrltsffiddsntvkfddittkkakeaiesnyfnklrtcsrmqdhfkeklaffypvyvkdkkdrpdddienlivlvknaiesvsylrnrtfhfkessllellkelddknsgqnkidysvaaefikrdienlydvfreqirslgiaeyykadmisdcflctcglefalyspknslmpafknvykrganlnkayirdkgpketgdqgqnsykaleeyreltwyievknndqsynayknllqliyyhaflpevrenealitdfinrtkewnrketeerintknnkkhknfdendditvntyryesipdyqgeslddylkvlqrkqmarakevnekeegnnnyiqfirdvvvwafgaylenklknyknelqpplskeniglndtlkelfpeekvkspfnikerfsistfidnkgkstdntsaeavktdgkedekdkknikrkdllcfylflrlldeneicklqhqfikyrcslkerrfpgnrtkleketellaeleelmelvrftmpsipeisakaesgydtmikkyflcdfiekkvfknpktsnlyyhsdsktpvtrkymallmrsaplhlykdifkgyylitkkecleyiklsniikdyqnslnelheqleriklksekqngkdslyldkkdfykykeyvenleqvarykhlqhkinfeslyrifrihvdiaarmvgytqdwerdmhflflcalvyngvleerrfeaifnnnddnndgrivkkiqnnlnnknrelvsmlcwnkklnknefgaiiwkrnpiahlnhftqteqnskssleslinslrillaydrkrqnavtktindlllndyhirikwegrvdegqiyfnikekedienepiihlkhlhkkdcyiyknsymfdkqkewicngikeevydksilkcigniflddyedknkssanpkht EreC2c2Eubacterium mlrrdkevkklynvfnqiqvgtkpkkwnndeklspeenerraqqknikmknykwrearectale cskyvessqriindvifysyrkaknklrymrknedilkkmqeaeklskfsggkledfvayt(SEQ ID lrkslvvskydtqefdslaamvvflecigknnisdhereivckllelirkdfskldpnvkgsqNO: 18) ganivrsvrnqnmivqpqgdrflfpqvyakenetvtnknvekeglnefllnyanlddekraeslrklrrildvyfsapnhyekdmditlsdniekekfnvwekhecgkketglfvdipdvlmeaeaenikldavvekrerkvindrvrkqniicyrytravvekynsneplffennainqywihhienaverilknckagklfklrkgylaekvwkdainlisikyialgkavynfalddiwkdkknkelgivderirngitsfdyemikahenlqrelavdiafsvnnlaravcdmsnlgnkesdfllwkrndiadklknkddmasysavlqffggksswdinifkdaykgkkkynyevrfiddlrkaiycarnenfhfktalvndekwntelfgkiferetefclnvekdrfysnnlymfyqvselrnmldhlysrsysraaqvpsynsvivrtafpeyitnvlgyqkpsydadtlgkwysacyyllkeiyynsflqsdralqlfeksvktlswddkkqqravdnfkdhfsdiksactslaqvcqiymteynqqnnqikkvrssndsifdqpvyqhykyllkkaianafadylknnkdlfgfigkpfkaneireidkeqflpdwtsrkyealcievsgKielqkwyivgkflnarslnlmvgsmrsyiqyvtdikrraasignelhvsvhdvekvekwvqvievesllasrtsnqfedyfndkddyarylksyvdfsnvdmpseysalvdfsneeqsdlyvdpknpkvnrnivhsklfaadhilrdivepvskdnieefysqkaeiayckikgkeitaeeqkavlkyqklknrvelrdiveygeiinellgqlinwsfmrerdllyfqlgfhydclrndskkpegyknikvdensikdailyqiigmyvngvtvyapekdgdklkeqcvkggvgvkvsafhryskylglnektlynagleifevvaehediinlrngidhfkyylgdyrsmlsiysevfdrfftydikyqknvlnllqnillrhnvivepilesgfktigeqtkpgaklsirsiksdtfqykvkggtlitdakderyletirkilyyaeneednlkksvvvtnadkyeknkesddqnkqkekknkdnkgkkneetksdaeknnnerlsynpfa nlnfklsnEbaC2C2 Eubacteriaceaemkiskeshkrtavavmedrvggvvyvpggsgidlsnnlkkrsmdtkslynvfnqiqagt bacteriumapseyewkdylseaenkkreaqkmiqkanyelrrecedyakkanlavsriifskkpkkif CHKCI004sdddiishmkkqrlskfkgrmedfvlialrkslvvstynqevfdsrkaatvflknigkknis (SEQ IDadderqikqlmaliredydkwnpdkdssdkkessgtkvirsiehqnmviqpeknklsls NO: 19)kisnvgkktktkqkekagldaflkeyaqidensrmeylkklrrlldtyfaapssyikgaavslpeninfsselnvwerheaakkvninfveipesllnaeqnnnkinkveqehsleqlrtdirrrnitcyhfanalaaderyhtlffenmamnqfwihhmenaverilkkenvgtlfldrigylsekvwkdmlnllsikyialgkavyhfalddiwkadiwkdasdknsgkindltlkgissfdyemvkaqedlqremavgvafstnnlarvtckmddlsdaesdfllwnkeairrhvkytekgeilsailqffggrslwdeslfekaysdsnyelkflddlkraiyaarnetfhfktaaidggswntrlfgslfekeaglclnveknkfysnnlvlfykqedlrvfldklygkecsraaqipsyntilprksfsdfmkqllglkepvygsaildqwysacyylfkevyynlflqdssakalfekavkalkgadkkqekavesfrkryweisknaslaeicqsyiteynqqnnkerkvrsandgmfnepiyqhykmllkealkmafasyikndkelkfvykpteklfevsqdnflpnwnsekyntlisevknspdlqkwyivgkfmnarmlnlllgsmrsylqyvsdiqkraaglgenqlhlsaenvgqvkkwiqvlevelllsvrisdkftdyfkdeeeyasylkeyvdfedsampsdysallafsnegkidlyvdasnpkvnrniiqaklyapdmvlkkvvkkisqdeckefnekkeqimqfknkgdevsweeqqkileyqklknrvelrdlseygelinellgqlinwsylrerdllyfqlgfhysclmneskkpdayktirrgtvsienavlyqiiamyingfpvyapekgelkpqcktgsagqkirafcqwasmvekkkyelynaglelfevvkehdniidlrnkidhfkyyqgndsilalygeifdrfftydmkyrnnvinhlqnillrhnviikpiiskdkkevgrgkmkdraaflleevssdrftykvkegerkidaknrlyletvrdilyfpnravndkgedviicskkaqdlnekkadrdknhdkskdtnqkkegknqeeksenkepysdrmtwkpfagikle C2c2 Lachno-mkiskvdhtrmavakgnqhrrdeisgilykdptktgsidfderfkklncsakilyhvfngi NK4A144spiraceaeaegsnkyknivdkvnnnldrvlftgksydrksiididtvlrnvekinafdristeereqiiddlbacteriumleiqlrkglrkgkaglrevlligagvivrtdkkqeiadfleildedfnktnqakniklsienqglNK4A144 vvspvsrgeerifdvsgaqkgksskkaqekealsaflldyadldknvrfeylrkirrlinlyf(SEQ ID yvknddvmslteipaevnlekdfdiwrdheqrkeengdfvgcpdilladrdvkksnskqNO: 20)vkiaerqlresireknikryrfsiktiekddgtyffankqisvfwihrienaverilgsindkklyrlrlgylgekvwkdilnflsikyiavgkavfnfamddlqekdrdiepgkisenavngltsfdyeqikademlqrevavnvafaannlarvtvdipqngekedillwnksdikkykknskkgilksilqffggastwnmkmfeiayhdqpgdyeenylydiiqiiyslrnksfhflaydhgdknwnreligkmiehdaervisverekfhsnnlpmfykdadlkkildllysdyagrasqvpafntylvrknfpefIrkdmgykvhfnnpevenqwhsavyylykeiyynlflrdkevknlfytslknirsevsdkkqklasddfasrceeiedrslpeicqiimteynaqnfgnrkvksqrvieknkdifrhykmlliktlagafslylkqerfafigkatpipyettdvknflpewksgmyasfyeeiknnldlqewyivgrflngrmlnqlagslrsyiqyaedierraaenrnklfskpdekieackkavrvldlcikistrisaeftdyfdseddyadylekylkyqddaikelsgssyaaldhfcnkddlkfdiyvnagqkpilqrnivmaklfgpdnilsevmekvtesaireyydylkkvsgyrvrgkcstekeqedllkfqrlknavefrdvteyaevinellgqliswsylrerdllyfqlgfhymclknksfkpaeyvdirrnngtiihnailyqivsmyingldfyscdkegktlkpietgkgvgskigqfikysqylyndpsykleiynaglevfenidehdnitdlrkyvdhfkyyaygnkmslldlyseffdrfftydmkyqknvvnvlenillrhfvifypkfgsgkkdvgirdckkeraqieiseqsltsedfmfklddkageeakkfparderylqtiakllyypneiedmnrfmkkgetinkkvqfnrkkkitrkqknnssnevlsstmgylfknikl C2c2 RNA-mtdqvrreevaageladtplaaaqtpaadaavaatpapaeavaptpeqavdqpattgese Chloro_binding apvttaqaaaheaepaeatgasftpvseqqpqkprrlkdlqpgmelegkvtsialygifvdagg protein S1vgvgrdglvhisemsdrridtpselvqigdtvkvwyksvdldarrisltmlnpsrgekprrChloroflexussrqsqpaqpqprrqevdreklaslkvgeivegvitgfapfgafadigvgkdglihiselseg aggregansrvekpedavkvgeryqfkvleidgegtrislslrraqrtqrmqqlepgqiiegtvsgiatfga (SEQ IDfvdigvgrdglvhisalaphrvakvedvvkvgdkvkvkvlgvdpqskrisltmrleeeqp NO: 21)attagdeaaepaeevtptrrgnlerfaaaaqtarersergersergerrerrerrpaqsspdtyivgedddesfegnatiedlltkfggsssrrdrdrrrrheddddeemerpsnrrqreairrtlqqi gydeC2c2 DemequinamdltwhallilfivallagfldtlagggglltvpallltgipplqalgtnklqssfgtgmatyqviDem_Aur aurantiacarkkrvhwrdvrwpmvwaflgsaagavavqfidtdalliiipvvlalvaayflfvpkshlpp (SEQ IDpeprmsdpayeativpiigaydgafgpgtgslyalsgvalraktivqstaiaktlnfatnfaal NO: 22)lvfafaghmlwtvgavmiagqligayagshmlfrvnplvlrvlivvmslgmlirvild C2c2Thalassospiramriikpygrshvegvatqeprrklrinsspdisrdipgfaqshdaliiaqwisaidkiatkpk Thal_Spsp. TSL5- pdkkptqaqinlrttlgdaawqhvmaenllpaatdpaireklhliwqskiapwgtarpqaTSL5 1 ekdgkptpkggwyerfcgvlspeaitqnvarqiakdiydhlhvaakrkgrepakqgess(SEQ ID nkpgkfkpdrkrglieeraesiaknalrpgshapcpwgpddqatyeqagdvagqiyaaaNO: 23) rdcleekkrrsgnrntssvqylprdlaakilyaqygrvfgpdttikaaldeqpslfalhkaikdcyhrlindarkrdilrilprnmaalfrlvraqydnrdinalirlgkvihyhaseqgksehhgirdywpsqqdiqnsrfwgsdgqadikrheafsriwrhiialasrtlhdwadphsqkfsgenddilllakdaieddvfkaghyerkedvlfgaqaslfcgaedfekailkqaitgtgnlrnatfhfkgkvrfekelqeltkdvpvevqsaiaalwqkdaegrtrqiaetlqavlaghflteeqnrhifaaltaamaqpgdvplprlrrvlarhdsicqrgrilplspcpdrakleespaltcqytvlkmlydgpfrawlaqqnstilnhyidstiartdkaardmngrklaqaekdlitsraadlprlsvdekmgdflarltaatatemrvqrgyqsdgenaqkqaafigqfecdvigrafadflnqsgfdfvlklkadtpqpdaaqcdvtaliapddisysppqawqqvlyfilhlvpvddashllhqirkwqvlegkekpaqiandvqsvlmlyldmhdakftggaalhgiekfaeffahaadfravfppqslqdqdrsiprrglreivrfghlpllqhmsgtvqithdnvvawqaartagatgmspiarrqkqreelhalavertarfrnadlqnymhalvdvikhrqlsaqvtlsdqvrlhrlmmgvlgrlvdyaglwerdlyfvvlallyhhgatpddvfkgqgkknladgqvvaalkpknrkaaapvgvfddldhygiyqddrqsirnglshfnmlrggkapdlshwvnqtrslvandrklknavaksviemlaregfdldwgiqtdrgqhilshgkirtrqaqhfqksrlhivkksakpdkndtvkirenlhgdamvervvqlfaaqvqkryditvekrldhlflkpqdqkgkngihthngwsktekkrrpsren rkgnhenC2c2 PseudobutyrmkfskeshrktavgvtesngiigllykdpinekekiedvvnqranstkrlfnlfgteatskdiPseudo_sp ivibrio sp.sraskdlakvvnkaignikgnkkfnkkeqitkglntkiiveelknvlkdekklivnkdiide OR37acsrllktsfrtaktkqavkmiltavlientnlskedeafvheyfvkklvneynktsvkkqip (SEQ IDvalsnqnmviqpnsvngtleisetkksketkttekdafraflrdyatldenrrhkmrlclrnl NO: 24)vnlyfygetsvskddfdewrdhedkkqndelfvkkivsiktdrkgnvkevldvdatidairtnniacyrralayanenpdvffsdtmlnkfwihhveneveriyghinnntgdykyqlgylsekvwkgiinylsikyiaegkavynyamnalakdnnsnafgkldekfvngitsfeyerikaeetlqrecavniafaanhlanatvdlnekdsdflllkhednkdtlgavarpnilrnilqffggksrwndfdfsgideiqllddlrkmiyslrnssfhfktenidndswntkligdmfaydfnmagnvqkdkmysnnvpmfystsdiekmldrlyaevherasqvpsfnsvfvrknfpdylkndlkitsafgvddalkwqsavyyvckeiyyndflqnpetftmlkdyvqclpididksmdqklksernahknfkeafatyckecdslsaicqmimteynnqnkgnrkvisartkdgdkliykhykmilfealknvftiyleknintygflkkpklinnvpaieeflpnyngrqyetlynriteetelqkwyivgrllnpkqvnqlignfrsyvqyvndvarrakqtgnnlsndniawdvkniiqifdvaklngvtsniledyfddgddyarylknfvdytnknndhsatllgdfcakeidgikigiyhdgtnpivnrniiqcklygatgiisdltkdgsilsvdyeiikkymqmqkeikvyqqkgicktkeeqqnlkkygelknivelrniidyseildelqgqlinwgylrerdlmyfqlgfhylclhneskkpvgynnagdisgavlyqivamytnglslidangkskknakasagakvgsfcsyskeirgvdkdtkedddpiylagvelfeninehqqcinlrnyiehfhyyakhdrsmldlysevfdrfftydmkytknvpnmmynillqhlvvpafefgssekrlddndeqtkpramftlreknglsseqftyrlgdgnstvklsargddylravasllyypdrapeglirdaeaedkfakinhsnpksdnrnnrgnfknpkvqwynnktkrk C2c2_Buty_ Butyrivibriomkiskvdhrktavkitdnkgaegfiyqdptrdsstmeqiisnrarsskvlfnifgdtkkskd sp sp.lnkytesliiyvnkaikslkgdkrnnkyeeiteslktervinaliqagneftcsenniedalnk YAB3001ylkksfrvgntksalkkllmaaycgyklsieekeeignyfvdklykeynkdtvlkytaksl (SEQ IDkhqnmvvqpdtdnhvflpsriagatqnkmsekealteflkayavldeekrhnlriilrklv NO: 25)nlyfyespdfiypennewkehddrknktetfvspvkvneekngktfvkidvpatkdlirlkniecyrrsvaetagnpityftdhniskfwihhienevekifallksnwkdyqfsvgyisekvwkeiinylsikyiaigkavynyaledikkndgtlnfgvidpsfydginsfeyekikaeetfqrevavyvsfavnhlssatvklseaqsdmlvinkndiekiaygntkrnilqffggqskwkefdfdryinpvnytdidflfdikkmvyslrnesfhftttdtesdwnknlisamfeyecrriStvqknkffsnnlplfygenslervlhklyddyvdrmsqvpsfgnvfvrkkfpdymkeigikhnlssednlklqgalyflykeiyynafissekamkifvdlynkldtnarddkgritheamahknfkdaishymthdcsladicqkimteynqqntghrkkqttysseknpeifrhykmilfmllqkamteyisseeifdfimkpnspktdikeeeflpqykscaydnlikliadnvelqkwyitarllsprevngligsfrsykqfvsdierraketnnslsksgmtvdvenitkvldlctklngrfsneltdyfdskddyavyvskfldfgfkidekfpaallgefcnkeengkkigiyhngtepilnsniiksklygitdvvsravkpvseklireylqqevkikpylengvcknkeeqaalrkyqelknriefrdiveyseiinelmgqlinfsylrerdlmyfqlgfhylclnnygakpegyysivndkrtikgailyqivamytyglpiyhyvdgtisdrrknkktvldtlnssetvgakikyfiyysdelfndslilynaglelfeninehenivnlrkyidhfkyyvsqdrslldiysevfdryftydrkykknymnlfsnimlkhfiitdfefstgektigekntakkecakvrikrgglssdkftykfkdakpielsakntefldgvarilyypenvvltdlvrnsevedekriekydrnhnssptrkdktykqdvkknynkktskafdsskldtksvgnnlsdnpvlkqflseskkkr C2c2_ Blautia sp.mkiskvdhvksgidqklssqrgmlykqpqkkyegkqleefivrnlsrkakalyqvfpvs Blautia_Marseille-gnskmekelqiinsfiknillrldsgktseeivgyintysvasqisgdhiqelvdqhlkeslrk spP2398 ytcvgdkriyvpdiivallkskfnsetlqydnselkilidfiredylkekqikqivhsiennst(SEQ ID plriaeingqkrlipanvdnpkksyifeflkeyaqsdpkgqesllqhmrylillylygpdkitNO: 26) ddyceeieawnfgsivmdneqlfseeasmliqdriyvnqqieegrqskdtakvkknkskyrmlgdkiehsinesvvkhyqeackaveekdipwikyisdhvmsvyssknrvdldklslpylakntwntwisfiamkyvdmgkgvyhfamsdvdkvgkqdnliigqidpkfsdgissfdyerikaeddlhrsmsgyiafavnnfaraicsdefrkknrkedvltvgldeiplydnykrkllqyfggasnwddsiidiiddkdlvacikenlyvarnvnfhfagsekvqkkqddileeivrketrdigkhyrkvfysnnvavfycdediiklmnhlyqrekpyqaqipsynkvisktylpdlifmllkgknrtkisdpsimnmfrgtfyfllkeiyyndflqasnlkemfceglknnvknkksekpyqnfmrrfeelenmgmdfgeicqqimtdyeqqnkqkkktatavmsekdkkirtldndtqkykhfrtllyiglreafiiylkdeknkewyeflrepvkreqpeekefvnkwklnqysdcselilkdslaaawyvvahfinqaqlnhligdiknyiqfisdidrrakstgnpvsesteiqieryrkilrvlefakffcgqitnyltdyyqdendfsthvghyvkfekknmepahalqafsnslyacgkekkkagfyydgmnpivnrnitlasmygnkkllenamnpvteqdirkyyslmaeldsvlkngavcksedeqknlrhfqnlknrielvdvltlselvndlvaqligwvyirerdmmylqlglhyiklyftdsvaedsylrtldleegsiadgavlyqiaslysfnlpmyvkpnkssvyckkhvnsvatkfdifekeycngdetvienglrlfeninlhkdmvkfrdylahfkyfakldesilelyskaydfffsyniklkksysyvltnyllsyfinaklsfstykssgnktvqhrttkisvvaqtdyftyklrsivknkngvesienddrrcevvniaardkefvdevcnvinynsdk C2c2_LeptotrichiamkitkidgishkkyikegklykstseenktderlselltirldtyiknpdnaseeenrirrenlLepto_sp_ sp.keffsnkvlylkdgilylkdrreknqlqnknyseediseydlknknnflvlkkillnedinseMarseille Marseille-eleifrndfekkldkinslkysleenkanyqkinennikkvegkskrnifynyykdsakrn P3007dyinniqeafdklykkedienlfflienskkhekykirecyhkiigrkndkenfatiiyeeiq (SEQ IDnvnnmkeliekvpnvselkksqvfykyylnkeklndenikyvfchfveiemskllkny NO: 27)vykkpsnisndkvkrifeyqslkklienkllnkldtyvrncgkysfylqdgeiatsdfivgnrqneaflrniigvsstayfslrniletenenditgrmrgktvknnkgeekyisgeidklydnnkqnevkknlkmfysydfnmnskkeiedffsnideaissirhgivhfnlelegkdiftflcnivpsqiskkmfhdeinekklklkifkqlnsanyfrylekykilnylnrtrfefvnknipfvpsftklysriddlknslgiywktpktnddnktkeitdaqiyllkniyygeflnyfmsnngnffeitkeiielnkndkrnlktgfyklqkfenlqektpkeylaniqslyminagnqdeeekdtyidfiqkifikgfmtylanngrlsliyigsdeetntslaekkqefdkflkkyeqnnnieipyeinefvreiklgkilkyterlnmfylilkllnhkeltnlkgslekyqsankeeafsdqlelinllnldnnrvtedfeleadeigkfldfngnkvkdnkelkkfdtnkiyfdgeniikhrafynikkygmlnllekisdeakykisieelknyskkkneieenhttgenlhrkyarprkdekftdedykkyekairniqqythlknkvefnelnllqsillrilhrlvgytsiwerdlrfrlkgefpenqyieeifnfdnsknvkykngqivekyinfykelykddtekisiysdkkvkelkkekkdlyirnyiahfnyipnaeisilemlenlrkilsydrklknaimksivdilkeygfvvtflciekdkkirieslkseevvhlkklklkdndkkkepiktyrnskelcklvkvmfeykmkekksen C2c2_ Bacteroidesmritkvkvkessdqkdkmvlihrkvgegtivldenladltapiidkykdksfelsllkqtiv Bacter-ihuae sekemnipkcdkctakerclsckgrekrlkevrgaiektigaviagrdiiprinifnedeicoides_ (SEQ IDwlikpkirneftfkdvnkqvvklnlpkvlveyskkndptiflayqqwiaaylknkkghik ihuaeNO: 28) ksilnnrvvidysdesklskrkqalelwgeeyetnqrialesyhtsynigelvtlipnpeeyvsdkgeirpafhyklknvlqmhqstvfgtneilcinpifnenraniqlsaynlevvkyfehyfpikkkkknlslnqaiyylkvetlkerlslqlenalrmnllqkgkikkhefdkntcsntlsqikrdeffvinlvemcafaannirnivdkeqvneilskkdlcnslskntidkelctkfygadfsqipvaiwamrgsvqqirneivhykaeaidkifalktfeyddmekdysdtpfkqylelsiekidsffieqlssndvinyyctedvnkllnkcklslrrtsipfapgfktiyelgchlqdssntyrighylmliggrvanstvtkaskaypayrfmlkliynhlflnkfldnhnkrffmkavafvlkdnrenarnkfqyafkeirmmnndesiasymsyihslsvqeqekkgdkndkvryntekfiekvfvkgfddflswlgvefilspnqeerdktvtreeyenlmikdrvehsinsnqeshiafftfcklldanhlsdlrnewikfrssgdkegfsynfaidiielclltvdrveqrrdgykeqtelkeylsffikgnesentvwkgfyfqqdnytpvlyspielirkygtlellkliivdedkitqgefeewqtlkkvvedkvtrrnelhqewedmknkssfsqekcsiyqklcrdidrynwldnklhlvhlrklhnlviqilsrmarfialwdrdfvlldasranddykilsffnfrdfinakktktddellaefgskiekknapfikaedvplmvecieakrsfyqkvffrnnlqvladrnfiahynyisktakcslfemiiklrtimyydrklrnavvksianvfdqngmvlqlslddshelkvdkviskrivhlknnnimtdqvpeeyykicrrllemkk C2c2_ Porphyromomefrdsiflcsllqkeiekaplcfaeklisggvfsyypserlkefvgnhpfslfrktmpfspgf Porph_nadaceae krvmksggnyqnanrdgrfydldigvylpkdgfgdeewnaryflmkliynqlflpyfadbacterium bacteriumaenhlfrecvdfvkrvnrdyncknnnseeqafidirsmredesiadylafiqsniiieenkk KH3CP3RAketnkegqinfnkfllqvfvkgfdsflkdrtelnflqlpelqgdgtrgddlesldklgavvav (SEQ IDdlkldatgidadlnenisfytfckildsnhlsrlrneiikyqsansdfshnedfdydriisiielcNO: 29) mlsadhvstndnesifpnndkdfsgirpylstdakvetfedlyvhsdaktpitnatmvlnwkygtdklferlmisdqdflvtekdyfvwkelkkdieekiklreelhslwvntpkgkkgakkkngrettgefseenkkeylevcreidryvnldnklhfvhlkrmhslliellgrfvgftylferdyqyyhleirsrrnkdagvvdkleynkikdqnkydkddffactflyekankvrnfiahfnyltmwnspqeeehnsnlsgaknssgrqnlkcsltelinelrevmsydrklknavtkavidlfdkhgmvikfrivnnnnndnknkhhlelddivpkkimhlrgiklkrqdgkpipiqtdsvdplycrmwkklldlkptpf C2c2_ Listeriamhdawaenpkkpqsdaflkeykacceaidtynwhknkativyvnelhhllidilgrlvg Listeria_riparia yvaiadrdfqcmanqylkssghtervdswintirknrpdyiekldifmnkaglfvsekngriparia (SEQIDrnyiahlnylspkhkysllylfeklremlkydrklknavtkslidlldkhgmcvvfanlknn NO: 30)khrlviaslkpkkietfkwkkik C2c2_ Insolitispir-mriirpygsstvaspspqdaqpirslqrqngtfdvaefsrrhpelvlaqwvamldkiirkpainsolitis_ illumpgknstalprptaeqrrlrqqvgaalwaemqrhtpvppelkavwdskvhpyskdnapat peregrinumperegrinum aktpshrgrwydrfgdpetsaatvaegvrrhlldsaqpfranggqpkgkgviehraltiqn(SEQ ID gtllhhhqsekagplpedwstyradelvstigkdarwikvaaslyqhygrifgpttpiseaqNO: 31)trpefvlhtavkayyrrlfkerklpaerlerllprtgealrhavtvqhgnrsladavrigkilhygwlqngepdpwpddaalyssrywgsdgqtdikhseaysrvwrraltaaqrtltswlypagtdagdilligqkpdsidrnflpllygdstrhwtrspgdvwlflkqtlenlrnssfhflalsaftshldgtcesepaeqqaaqalwqddrqqdhqqvflslraldattylptgplhrivnavqstdatlplprfrrvvtraantrlkgfpvepvnrrtmeddpllrcrygvlkllyergfrawletrpsiascldqslkrstkaaqtingknspqgveilsratkllqaegggghgihdlfdrlyaataremrvqvgyhhdaeaarqqaefiedlkcevvarafcaylktlgiqgdtfrrqpeplptwpdlpdlpsstigtaqaalysvlhlmpvedvgsllhqlrrwlvalqarggedgtaitatipllelylnrhdakfsgggagtglrwddwqvffdcqatfdrvfppgpaldshrlplrglrevlrfgrvndlaaligqdkitaaevdrwhtaeqtiaaqqqrrealheqlsrkkgtdaevdeyralvtaiadhrhltahvtlsnvvrlhrlmttvlgrlvdygglwerdltfvtlyeahrlgglrnllsesrvnkfldgqtpaalskknnaeengmiskylgdkarrqirndfahfnmlqqgkktinitdeinnarklmandrklknaitrsvttllqqdgldivwtmdashrltdakidsrnaihlhkthnranireplhgksycrwvaalfgatstpsatkksdkir

In an embodiment of the invention, there is provided effector proteinwhich comprises an amino acid sequence having at least 80% sequencehomology to the wild-type sequence of any of Leptotrichia shahii C2c2,Lachnospiraceae bacterium MA2020 C2c2, Lachnospiraceae bacterium NK4A179C2c2, Clostridium aminophilum (DSM 10710) C2c2, Carnobacteriumgallinarum (DSM 4847) C2c2, Paludibacter propionicigenes (WB4) C2c2,Listeria weihenstephanensis (FSL R9-0317) C2c2, Listeriaceae bacterium(FSL M6-0635) C2c2, Listeria newyorkensis (FSL M6-0635) C2c2,Leptotrichia wadei (F0279) C2c2, Rhodobacter capsulatus (SB 1003) C2c2,Rhodobacter capsulatus (R121) C2c2, Rhodobacter capsulatus (DE442) C2c2,Leptotrichia wadei (Lw2) C2c2, or Listeria seeligeri C2c2.

In an embodiment of the invention, the effector protein comprises anamino acid sequence having at least 80% sequence homology to a Type VIeffector protein consensus sequence including but not limited to aconsensus sequence described herein

According to the invention, a consensus sequence can be generated frommultiple C2c2 orthologs, which can assist in locating conserved aminoacid residues, and motifs, including but not limited to catalyticresidues and HEPN motifs in C2c2 orthologs that mediate C2c2 function.One such consensus sequence, generated from the 33 orthologs mentionedabove using Geneious alignment is:

(SEQ ID NO: 32) mkiskvxxxvxkkxxxgklxkxvnernrxakrlsnxlbkyixxidkixkkexxkkfxaxeeitlklnqxxxbxlxkaxxdlrkdnxysxjkkilhnedinxeexellindxleklxkiesxkysyqkxxxnyxmsvqehskksixrixesakrnkealdkflkeyaxldprmexlaklrkllelyfyfkndxixxeeexnvxxhkxlkenhpdfvexxxnkenaelnxyaiexkkjlkyyfpxkxaknsndkifekqelkkxwihqjenaverillxxgkvxyklqxgylaelwkirineifikyixvgkavaxfalmxxkbendilggkixkklngitsfxyekikaeeilqrexavevafaanxlyaxdlxxirxsilqffggasnwdxflffhfatsxisdkkwnaelixxkkjglvireklysnnvamfyskddlekllnxlxxfxlrasqvpsfkkvyvrxbfpqnllkkfndekddeaysaxyyllkeiyynxflpyfsannxfffxvknlvlkankdkfxxafxdiremnxgspieylxxtqxnxxnegrkkeekexdfikfllqifxkgfddylknnxxfilkfipeptexieixxelqawyivgkflnarkxnllgxfxsylkllddielralrnenikyqssnxekevlexcleligllsldlndyfbdexdfaxyjgkxldfekkxmkdlaelxpydqndgenpivnrnixlakkygtlnllekjxdkvsekeikeyyelkkeieeyxxkgeelheewxqxknrvexrdileyxeelxgqiinynxlxnkvllyfqlglhyllldilgrlvgytgiwerdaxlyqiaamyxnglpeyixxkkndkykdgqivgxkinxfkxdkkxlynaglelfenxnehknixirnyiahfnylskaessllxysenlrxlfsydrklknavxkslinillrhgmvlkfldgtdkksvxirsxkkixhlksiakklyypevxvskeycklvkxllkyk

In another non-limiting example, a sequence alignment tool to assistgeneration of a consensus sequence and identification of conservedresidues is the MUSCLE alignment tool (www.ebi.ac.uk/Tools/msa/muscle/).For example, using MUSCLE, the following amino acid locations conservedamong C2c2 orthologs can be identified in Leptotrichia wadei C2c2:K2;K5; V6; E301; L331; I335; N341; G351; K352; E375; L392; L396; D403;F446; I466; I470; R474 (HEPN); H475; H479 (HEPN), E508; P556; L561;I595; Y596; F600; Y669; I673; F681; L685; Y761; L676; L779; Y782; L836;D847; Y863; L869; I872; K879; 1933; L954; I958; R961; Y965; E970; R971;D972; R1046 (HEPN), H1051 (HEPN), Y1075; D1076; K1078; K1080; 11083;11090.

An exemplary sequence alignment of HEPN domains showing highly conservedresidues is shown in FIG. 50

In certain example embodiments, the RNA-targeting effector protein is aType VI-B effector protein, such as Cas13b and Group 29 or Group 30proteins. In certain example embodiments, the RNA-targeting effectorprotein comprises one or more HEPN domains. In certain exampleembodiments, the RNA-targeting effector protein comprises a C-terminalHEPN domain, a N-terminal HEPN domain, or both. Regarding example TypeVI-B effector proteins that may be used in the context of thisinvention, reference is made to U.S. application Ser. No. 15/331,792entitled “Novel CRISPR Enzymes and Systems” and filed Oct. 21, 2016,International Patent Application No. PCT/US2016/058302 entitled “NovelCRISPR Enzymes and Systems”, and filed Oct. 21, 2016, and Smargon et al.“Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentiallyregulated by accessory proteins Csx27 and Csx28” Molecular Cell, 65,1-13 (2017); dx.doi.org/10.1016/j.molcel.2016.12.023, and U.S.Provisional application No. to be assigned, entitled “Novel Cas13bOrthologues CRISPR Enzymes and System” filed Mar. 15, 2017. Inparticular embodiments, the Cas13b enzyme is derived from Bergeyellazoohelcum. In certain other example embodiments, the effector proteinis, or comprises an amino acid sequence having at least 80% sequencehomology to any of the sequences listed in Table 6.

TABLE 6 Bergeyella  1menktsignniyynpfkpqdksyfagyfnaamentdsvfrelgkrlkgkeytsenffdaifkenizoohelcum (SEQ IDslveyeryvkllsdyfpmarlldkkevpikerkenfkknfkgiikavrdlrnfythkehgeveitdeNO: 33)ifgvldemlkstvltvkkkkvktdktkeilkksiekqldilcqkkleylrdtarkieekrrnqrergekelvapfkysdkrddliaaiyndafdvyidkkkdslkesskakyntksdpqqeegdlkipiskngvvfilslfltkqeihafkskiagfkatvideatvseatvshgknsicfmatheifshlaykklkrkvrtaeinygeaenaeqlsvyaketlmmqmldelskvpdvvyqnlsedvqktfiedwneylkenngdvgtmeeeqvihpvirkryedkfnyfairfldefaqfpflrfqvhlgnylhdsrpkenlisdrrikekitvfgrlselehkkalfikntetnedrehyweifpnpnydfpkenisvndkdfpiagsildrekqpvagkigikvkllnqqyvsevdkavkahqlkqrkaskpsiqniieeivpinesnpkeaivfggqptaylsmndihsilyeffdkwekkkeklekkgekelrkeigkelekkivgkiqaqiqqiidkdtnakilkpyqdgnstaidkeklikdlkqeqnilqklkdeqtvrekeyndfiayqdknreinkvrdrnhkqylkdnikrkypeaparkevlyyrekgkvavwlandikrfmptdfknewkgeqhsllqkslayyeqckeelknllpekvfqhlpfklggyfqqkylyqfytcyldkfleyisglvqqaenfksenkvfkkvenecfkfikkqnythkeldarvqsilgypiflergfmdekptiikgktfkgnealfadwfryykeyqnfqtfydtenyplvelekkqadrkrktkiyqqkkndvifilmakhifksvfkqdsidqfsledlyqsreerlgnqerarqtgerntnyiwnktvdlklcdgkitvenvklknvgdfikyeydqrvqaflkyeeniewqaflikeskeeenypyvvereiegyekvrreellkevhlieeyilekvkdkeilkkgdnqnfkyyilngllkqlknedvesykvfnintepedvninqlkqeatdleqkafvltyirnkfahnqlpkkefwdycqekygkiekektyaeyfaevflckekealik Prevotella  2meddkkttdsiryelkdkhfwaafinlarhnvyitvnhinkileegeinrdgyettikntwneikdiintermedia (SEQ IDnkkdrlskliikhfpfleaatyrinptdttkqkeekqaeaqsleslrksffvflyklrdlrnhyshykhNO: 34)skslerpkfeegllekmynifnasirlykedyqynkdinpdedflchldrteeefnyyftkdnegnitesgllffvslflekkdaiwmqqklrgfkdnrenkkkmtnevfcrsrmllpklrlqstqtqdwilldmlnelircpkslyerlreedrekfrvpieiadedydaeqepflcntivrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkqigdykeshhlthklygferiqeftkqnrpdewrkfvktfnsfetskepyipettphyhlenqkigirfrndndkiwpslktnseknekskykldksfqaeaflsvhellpmmfyylllktentdndneietkkkenkndkqekhkieeiienkiteiyalydtfangeiksideleeyckgkdieighlpkqmiailkdehkvmateaerkqeemlvdvqkslesldnqineeienverknsslksgkiaswlyndmmrfqpvqkdnegkpinnskansteyqllqrtlaffgseherlapyfkqtkliessnphpflkdtewekcnnilsfyrsyleakknfleslkpedweknqyflklkepktkpktivqgwkngfnlprgiftepirkwfmkhrenitvaelkrvglvakviplffseeykdsvqpfynyhfnvgninkpdeknfinceerrellrkkkdefkkmtdkekeenpsylefkswnkferelrlvrnqdivtwllcmelfnkkkikelnvekiylknintnttkkeknteekngeeknikeknnilnrimpmrlpikvygrenfsknkkkkirrntfftvyieekgtkllkqgnfkalerdrrlgglfsfvktpskaesksntisklrveyelgeyqkarieiikdmlalektlidkynsldtdnfnkmltdwlelkgepdkasfqndvdlliavrnafshnqypmrnriafaninpfslssantseekglgianqlkdkthktiekiieiekpietke Prevotella 3 mqkqdklfvdrkknaifafpkyitimenkekpepiyyeltdkhfwaaflnlarhnvyttinhinrrbuccae (SEQ IDleiaelkddgymmgikgswneqakkldkkvrlrdlimkhfpfleaaayemtnskspnnkeqre NO: 35)keqsealslnnlknvlfifleklqvlrnyyshykyseespkpifetsllknmykvfdanvrlvkrdymhhenidmqrdfthlnrkkqvgrtkniidspnfhyhfadkegnmtiagllffvslfldkkdaiwmqkklkgflcdgrnlreqmtnevfcrsrislpklklenvqtkdwmqldmlnelvrcpkslyerlrekdresfkvpfdifsddynaeeepfkntivrhqdrfpyfvlryfdlneifeqlrfqidlgtyhfsiynkrigdedevrhlthhlygfariqdfapqnqpeewrklykdldhfetsqepyisktaphyhlenekigikfcsahnnlfpslqtdktengrskfnlgtqftaeaflsvhellpmmfyyllltkdysrkesadkvegiirkeisniyaiydafanneinsiadltrrlqntnilqghlpkqmisilkgrqkdmgkeaerkigemiddtqrrldllckqtnqkirigkrnagllksgkiadwlyndmmrfqpvqkdqnnipinnskansteyrmlqralalfgsenfrlkayfnqmnlvgndnphpflaetqwehqtnilsfyrnylearkkylkglkpqnwkqyqhflilkvqktnrntivtgwknsfnlprgiftqpirewfekhnnskriydqilsfdrygfvakaiplyfaeeykdnvqpfydypfnignrlkpkkredkkervelwqknkelfknypsekkktdlayldflswkkferelrliknqdivtwlmflcelfnmatveglkigeihlrdidtntaneesnnilnrimpmklpvktyetdnkgnilkerplatfyieetetkvlkqgnfkalvkdrringlfsfaettdlnleehpisklsvdlelikyqttrisifemtlglekklidkystlptdsfrnmlerwlqckanrpelknyvnsliavrnafshnqypmydatlfaevkkftlfpsvdtkkielniapqlleivgkaikeieksenknPorphyromonas  4mntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllcdhllsvgingivalis (SEQ IDdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslldflrndfshnrldgttfNO: 36)ehlevspdissfitgtyslacgraqsrfavffkpddfvlaknrkeqlisvadgkecltvsgfafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsensldeesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmdqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelrlldpssghpflsatmetahrytegfykcylekkrewlakifyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskvmellkykdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvrdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpi ndlBacteroides  5mesiknsqkstgktlqkdppyfglylnmallnyrkvenhirkwlgdvallpeksgfhsllttdnlsspyogenes (SEQ IDakwtrfyyksrkflpflemfdsdkksyenrretaecldtidrqkissllkevygklqdirnafshyhiNO: 37)ddqsvkhtaliissemhrfienaysfalqktrarftgvfvetdflqaeekgdnkkffaiggnegiklkdnalifliclfldreeafkflsratgflcstkekgflavretfcalccrqpherllsvnpreallmdmlnelnrcpdilfemldekdqksflpllgeeeqahilenslndelceaiddpfemiaslskrvryknrfpylmlryieeknllpfirfridlgclelasypkkmgeennyersvtdhamafgrltdfhnedavlqqitkgitdevrfslyapryaiynnkigfvrtsgsdkisfptlkkkggeghcvaytlqntksfgfisiydlrkilllsfldkdkaknivsglleqcekhwkdlsenlfdairtelqkefpvplirytlprskggklvsskladkqekyeseferrkeklteilsekdfdlsqiprrmidewlnvlptsrekklkgyvetlkldcrerlrvfekrekgehplpprigematdlakdiirmvidqgvkqritsayyseiqrclaqyagddnrrhldsiirelrlkdtknghpflgkvlrpglghteklyqryfeekkewleatfypaaspkrvprfvnpptgkqkelpliirnlmkerpewrdwkqrknshpidlpsqlfeneicrllkdkigkepsgklkwnemfklywdkefpngmqrfyrckrrvevfdkvveyeyseeggnykkyyealidevvrqkissskeksklqvedltlsvrrvflcrainekeyqlrllceddrllfmavrdlydwkeaqldldkidnmlgepvsysqviqleggqpdavikaecklkdvsklmrycydgrvkglmpyfanheatqeqvemelrhyedhrrrvfnwvfaleksvlknekliffyeesqggcehrrcidalrkaslvseeeyeflvhirnksahnqfpdleigklppnvtsgfceciwskykaiicriipfidperrffgklleqk Alistipes  6msneigafrehqfayapgnekqeeatfatyfnlalsnvegmmfgevesnpdkieksldtlppail sp.(SEQ IDrqiasfiwlskedhpdkaysteevkvivtdlvrrlcfyrnyfshcfyldtqyfysdelvdttaigeklpZOR0009 NO: 38)ynfhhfitnrlfryslpeitlfrwnegerkyeilrdgliffcclflkrgqaerflnelrffkrtdeegrikrtiftkyctreshkhigieeqdflifqdiigdlnrvpkvcdgvvdlskeneryiknretsnesdenkaryrllirekdkfpyylmryivdfgvlpcitfkqndystkegrgqfhyqdaavaqeercynfvvrngnvyysympqaqnvvriselqgtisveelrnmvyasingkdvnksveqylyhlhllyekiltisgqtikegrvdvedyrplldklllrpasngeelrrelrkllpkrvcdllsnrfdcsegvsavekrlkaillrheqlllsqnpalhidkiksvidylylffsddekfrqqptekahrglkdeefqmyhylvgdydshplalwkeleasgrlkpemrkltsatslhglymlclkgtvewcrkqlmsigkgtakveaiadrvglklydklkeytpeqlerevklvvmhgyaaaatpkpkaqaaipskltelrfysflgkremsfaafirqdkkaqklwlrnfytveniktlqkrqaaadaackklynlvgevervhtndkvlvlvaqryrerllnvgskcavtldnperqqkladvyevqnawlsirfddldftlthvnlsnlrkaynliprkhilafkeyldnrvkqklceecrnvrrkedlctccsprysnitswlkenhsessiereaatmmlldverkllsfllderrkaiieygkfipfsalvkecrladaglcgirndvlhdnvisyadaigklsayfpkeaseaveyirrtkevreqrreelmanssq Prevotella  7amskeckkqrqekkrrlqkanfsisltgkhvfgayfnmartnfvktinyilpiagvrgnysenqink sp.(SEQ IDmlhalfliqagrneeltteqkqwekklrinpeqqtkfqkllflchfpvlgpmmadvadhkaylnk MA2016NO: 39)kkstvqtedetfamlkgvsladcldiiclmadtltecrnfythkdpynkpsqladqylhqemiakkldkvvvasrrilkdreglsvnevefltgidhlhqevlkdefgnakvkdgkvmktfveyddfyflcisgkrlvngytvttkddkpvnvntmlpalsdfgllyfcvlflskpyaklfidevrlfeyspfddkenmimsemlsiyrirtprlhkidshdskatlamdifgelrrcpmelynlldknagqpffhdevkhpnshtpdvskrlryddrfptlalryidetelfkrirfqlqlgsfrykfydkencidgrvrvrriqkeingygrmqevadkrmdkwgdliqkreersvkleheelyinldqfledtadstpyvtdrrpaynihanriglywedsqnpkqykvfdengmyipelvvtedkkapikmpaprcalsvydlpamlfyeylreqqdnefpsaeqviieyeddyrkffkavaegklkpfkrpkefrdflkkeypklrmadipkklqlflcshglcynnkpetvyerldrltlqhleerelhiqnrlehyqkdrdmignkdnqygkksfsdvrhgalarylaqsmmewqptklkdkekghdkltglnynyltaylatyghpqvpeegftprtleqvlinahliggsnphpfinkvlalgnrnieelylhyleeelkhirsriqslssnpsdkalsalpfihhdrmryhertseemmalaaryttiqlpdglftpyileilqkhytensdlqnalsqdvpvklnptcnaaylitlfyqtvlkdnaqpfylsdktytrnkdgekaesfsfkrayelfsvinnnkkdtfpfemiplfltsdeigerlsaklldgdgnpvpevgekgkpatdsqgntiwkrriysevddyaekltdrdmkisfkgeweklprwkqdkiikrrdetrrqmrdellqrmpryirdikdnertlrryktqdmvlfllaekmftniiseqssefnwkqmrlskvcneaflrqtltfrvpvtvgettiyveqenmslknygefyrfltddrlmsllnnivetlkpnengdlvirhtdlmselaaydqyrstifmliqsienliitnnavlddpdadgfwvredlpkrnnfasllelinqlnnveltdderkllvairnafshnsynidfslikdvkhlpevakgilqhlqsmlgveitk Prevotella 7b mskeckkqrqekkrrlqkanfsisltgkhvfgayfnmartnfvktinyilpiagvrgnysenqinksp. (SEQ IDmlhalfliqagrneeltteqkqwekkhinpeqqtkfqkllflchfpvlgpmmadvadhkaylnk MA2016NO: 40)kkstvqtedetfamlkgvsladcldiiclmadtltecrnfythkdpynkpsqladqylhqemiakkldkvvvasrrilkdreglsvnevefltgidhlhqevlkdefgnakvkdgkvmktfveyddfyfkisgkrlvngytvttkddkpvnvntmlpalsdfgllyfcvlflskpyaklfidevrlfeyspfddkenmimsemlsiyrirtprlhkidshdskatlamdifgebrcpmelynlldknagqpffhdevkhpnshtpdvskrlryddrfptlalryidetelfkrirfqlqlgsfrykfydkencidgrvrvrriqkeingygrmqevadkrmdkwgdliqkreersvkleheelyinldqfledtadstpyvtdrrpaynihanriglywedsqnpkqykvfdengmyipelvvtedkkapikmpaprcalsvydlpamlfyeylreqqdnefpsaeqviieyeddyrkffkavaegklkpfkrpkefrdflkkeypklrmadipkklqlflcshglcynnkpetvyerldrltlqhleerelhiqnrlehyqkdrdmignkdnqygkksfsdvrhgalarylaqsmmewqptklkdkekghdkltglnynyltaylatyghpqvpeegftprtleqvlinahliggsnphpfinkvlalgnrnieelylhyleeelkhirsriqslssnpsdkalsalpfihhdrmryhertseemmalaaryttiqlpdglftpyileilqkhytensdlqnalsqdvpvklnptcnaaylitlfyqtvlkdnaqpfylsdktytrnkdgekaesfsfkrayelfsvinnnkkdtfpfemiplfltsdeigerlsaklldgdgnpvpevgekgkpatdsqgntiwkrriysevddyaekltdrdmkisfkgeweklprwkqdkiikrrdetrrqmrdellqrmpryirdikdnertlrryktqdmvlfllaekmftniiseqssefnwkqmrlskvcneaflrqtltfrvpvtvgettiyveqenmslknygefyrfltddrlmsllnnivetlkpnengdlvirhtdlmselaaydqyrstifmliqsienliitnnavlddpdadgfwvredlpkrnnfasllelinqlnnveltdderkllvairnafshnsynidfslikdvkhlpevakgilqhlqsmlgveitk Riemerella 8 mekpllpnvytlkhkffwgaflniarhnafitichineqlglktpsnddkivdvvcetwnnilnndanatipestifer (SEQ IDhdllkksqltelilkhfpfltamcyhppkkegkkkghqkeqqkekeseaqsqaealnpskliealeNO: 41)ilvnqlhslrnyyshykhkkpdaekdifkhlykafdaslrmvkedykahftvnitrdfahlnrkgknkqdnpdfnryrfekdgfftesgllfftnlfldkrdaywmlkkvsgfkashkqrekmttevfcrsrillpkblesrydhnqmlldmlselsrcpkllyeklseenkkhfqveadgfldeieeeqnpfkdtlirhqdrfpyfalryldlnesfksirfqvdlgtyhyciydkkigdeqekrhltrtllsfgrlqdfteinrpqewkaltkdldyketsnqpfiskttphyhitdnkigfrlgtskelypsleikdganriakypynsgfvahafisvhellplmfyqhltgksedllketvrhiqriykdfeeerintiedlekanqgflplgafpkqmlgllqnkqpdlsekakikiekliaetkllshrintklksspklgkrrekliktgvladwlvkdfmrfqpvaydaqnqpiksskanstefwfirralalyggeknrlegyfkqtnligntnphpflnkfnwkacrnlvdfyqqylegekfleaiknqpwepyqyclllkipkenrknlvkgweqggislprglfteairetlsedlmlskpirkeikkhgrvgfisraitlyfkekyqdkhqsfynlsykleakapllkreehyeywqqnkpqsptesqrlelhtsdrwkdyllykrwqhlekklrlyrnqdvmlwlmtleltknhfkelnlnyhqlklenlavnvqeadaklnpinqtlpmvlpvkvypatafgevqyhktpirtvyireehtkalkmgnfkalvkdrringlfsfikeendtqkhpisqlrlrreleiyqslrvdafketlsleekllnkhtslsslenefralleewkkeyaassmvtdehiafiasvrnafchnqypfykealhapiplftvaqptteekdglgiaeallkvlreyceivksqi Prevotella  9meddkkttgsisyelkdkhfwaaflnlarhnvyitinhinklleireidndekvldiktlwqkgnkaurantiaca (SEQ IDdlnqkarlrelmtkhfpfletaiytknkedkkevkqekqaeaqsleslkdclflfldklqearnyysNO: 42) hykysefskepefeegllekmynifgnniqlvindyqhnkdinpdedfkhldrkgqfkysfadnegnitesgllffvslflekkdaiwmqqklngfkdnlenkkkmthevfcrsrilmpklrlestqtqdwilldmlnelircpkslyerlqgddrekfkvpfdpadedynaeqepfkntlirhqdrfpyfvlryfdyneifknlrfqidlgtyhfsiykkliggqkedrhlthklygferiqefakqnrpdewkaivkdldtyetsnkryisettphyhlenqkigirfrngnkeiwpslktndennekskykldkqyqaeaflsvhellpmmfyylllkkekpnndeinasivegfikreirnifklydafangeinniddlekycadkgipkrhlpkqmvailydehkdmvkeakrkqkemvkdtkkllatlekqtqkekeddgrnvkllksgeiarwlyndmmrfqpvqkdnegkpinnskansteyqmlqrslalynneekptryfrqvnliesnnphpflkwtkweecnniltfyysyltkkieflnklkpedwkknqyflklkepktnretivqgwkngfnlprgiftepirewfkrhqnnskeyekvealdrvglvtkviplffkeeyfkdkeenfkedtqkeindcvqpfynfpynvgnihkpkekdflhreerielwdkkkdkfkgykekikskkltekdkeefrsylefqswnkferelrlvrnqdivtwllckelidklkidelnieelkklrinnidtdtakkeknnilnrvmpmelpvtvyeiddshkivkdkplhtiyikeaetkllkqgnfkalvkdrringlfsfvktnseaeskrnpisklrveyelgeyqearieiiqdmlaleeklinkykdlptnkfsemlnswlegkdeadkarfqndvdfliavrnafshnqypmhnkiefanikpfslytannseekglgianqlkdktkettdkikkiekpi etkePrevotella 10medkpfwaaffnlarhnvyltvnhinklldleklydegkhkeiferedifnisddvmndansngsaccharolytica (SEQ IDkkrkldikkiwddldtdltrkyqlrelilkhfpfiqpaiigaqtkerttidkdkrststsndslkqtgegNO: 43)dindllslsnyksmffrllqileqlrnyyshvkhsksatmpnfdedllnwmryifidsvnkvkedyssnsvidpntsfshliykdeqgkikperypftskdgsinafgllffvslflekqdsiwmqkkipgfkkasenymkmtnevfcrnhillpkirletvydkdwmlldmlnevvrcplslykrltpaaqnkfkvpekssdnanrqeddnpfsrilvrhqnrfpyfvlrffdlnevfttlrfqinlgcyhfaickkqigdkkevhhlirtlygfsrlqnftqntrpeewntivkttepssgndgktvqgvplpyisytiphyqienekigikifdgdtavdtdiwpsvstekqlnkpdkytltpgfkadvflsvhellpmmfyygillcegmlktdagnavekvlidtrnaifnlydafvqekintitdlenylqdkpilighlpkqmidllkghqrdmlkaveqkkamlikdterrlklldkqlkqetdvaakntgtllkngqiadwlvndmmrfqpvkrdkegnpincskansteyqmlqrafafyatdscrlsryftqlhlihsdnshlflsrfeydkqpnliafyaaylkakleflnelqpqnwasdnyflllrapkndrqklaegwkngfnlprglftekiktwfnehktivdisdcdifknrvgqvarlipvffdkkfkdhsqpfyrydfnvgnvskpteanylskgkreelfksyqnkflainipaektkeyreyknfslwkkferelrliknqdiliwlmcknlfdekikpkkdilepriaysyikldslqtntstagslnalakvvpmtlaihidspkpkgkagnnekenkeftvyikeegtkllkwgnflctlladrrikglfsyiehddidlkqhpltkrrvdleldlyqtcridifqqtlgleaqlldkysdlntdnfyqmligwrkkegiprnikedtdflkdvrnafshnqypdskkiafrrirkfnpkelileeeeglgiatqmykevekvvnrikrielfd HMPREF9712_ 11mkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdngnlknyii03108 (SEQ IDhvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltlsqrirqfremlislvtavdqlrnfyth[Myroides NO: 44)yhhsdivienkvldflnssfvstalhvkdkylktdktkeflketiaaeldilieaykkkqiekkntrfkodoratimimusankredilnaiyneafwsfindkdkdkdketvvakgadayfeknhhksndpdfalnisekgivy CCUGllsffltnkemdslkanitgfkgkvdresgnsikymatqriysfhtyrglkqkirtseegvketllmq10230] midelskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvihpvirkryedrfnyfairfldeffdfptlrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakasyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnyksekplvftgqpiaylsmndihsmlfslltdnaelkktpeeveaklidqigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmflceskskwkgyqhtelqklfayfdtsksdlelilsnmvmvkdypielidlykksrtivdflnkylearleyienvitrvknsigtpqflavrkecftflkksnytvvsldkqverilsmplfiergfmddkptmlegksykqhkekfadwfvhykensnyqnfydtevyeittedkrekakvtkkikqqqkndvifimmvnymleevlklssndrlslnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlcdglvhidnvklkdignfrkyendsrvkefltyqsdivwsaylsnevdsnklyvierqldnyesirskellkevqeiecsvynqvankeslkqsgnenfkqyvlqgllpigmdvremlilstdvkfkkeeiiqlgqageveqdlysliyirnkfahnqlpikeffdfcennyrsisdneyyaeyymeifrsikekyan Prevotella 12meddkkttdsiryelkdkhfwaaflnlarhnvyitvnhinkileedeinrdgyentlenswneikdintermedia (SEQ IDinkkdrlskliikhfpfleattyrqnptdttkqkeekqaeaqsleslkksffvflyklrdlrnhyshykNO: 45)hskslerpkfeedlqnkmynifdvsiqfvkedykhntdinpkkdflchldrkrkgkfhysfadnegnitesgllffvslflekkdaiwvqkklegflccsnksyqkmtnevfcrsrmllpklrlestqtqdwilldmlnelircpkslyerlqgvnrkkfyvsfdpadedydaeqepflcntivrhqdrfpyfalryfdynevfanlrfqidlgtyhfsiykkliggqkedrhlthklygferiqefdkqnrpdewkaivkdsdifickkeekeeekpyisettphyhlenkkigiafknhniwpstqteltnnkrkkynlgtsikaeaflsvhellpmmfyylllktentkndnkvggkketkkqgkhkieaiieskikdiyalydafangeinsedelkeylkgkdikivhlpkqmiailknehkdmaekaeakqekmklatenrlktldkqlkgkiqngkrynsapksgeiaswlyndmmrfqpvqkdengeslnnskansteyqllqrtlaffgseherlapyfkqtkliessnphpflndtewekcsnilsfyrsylkarknfleslkpedweknqyflmlkepktnretivqgwkngfnlprgfftepirkwfmehwksikvddlkrvglvakvtplffsekykdsvqpfynypfnvgdynkpkeedflhreerielwdkkkdkflcgykakkkfkemtdkekeehrsylefqswnkferelrlyrnqdivtwllctelidklkidelnikelkklrlkdintdtakkeknnilnrvmpmelpvtvykynkggyiiknkplhtiyikeaetkllkqgnfkalvkdrringlfsfvktpseaesesnpisklrveyelgkyqnarldiiedmlalekklidkynsldtdnfhnmltgwlelkgeakkarfqndvklltavrnafshnqypmydenlfgnierfslsssniieskgldiaaklkeevskaakkiqneednkkeketCapnocyto- 13mkniqrlgkgnefspfkkedkfyfggflnlannniedffkeiitrfgivitdenkkpketfgekilnephaga (SEQ IDifkkdisivdyekwvnifadyfpftkylslyleemqfknrvicfrdvmkellktvealrnfythydcanimorsus NO: 46)hepikiedrvfyfldkvildvsltvknkylktdktkeflnqhigeelkelckqrkdylvgkgkridkeseiingiynnaflcdfickrekqddkenhnsvekilcnkepqnkkqkssatvwelcskssskyteksfpnrendkhclevpisqkgivfllsifinkgeiyaltsnikgflcakitkeepvtydknsirymathrmfsflaykglkrkirtseinynedgqasstyeketlmlqmldelnkvpdvvyqnlsedvqktfiedwneylkenngdygtmeeeqvihpvirkryedkfnyfairfldefaqfptlrfqvhlgnylcdkrtkqicdttterevkkkitvfgrlselenkkaiflnereeikgwevfpnpsydfpkenisvnykdfpivgsildrekqpvsnkigirvkiadelqreidkaikekklrnpknrkanqdekqkerlvneivstnsneqgepvvfigqptaylsmndihsvlyeflinkisgealetkivekietqikqiigkdattkilkpytnansnsinrekllrdleqeqqilktlleeqqqrekdkkdkkskrkhelypsekgkvavwlandikrfmpkafkeqwrgyhhsllqkylayyeqskeelknllpkevflchfpfklkgyfqqqylnqfytdylkrrlsyvnelllniqnfkndkdalkatekecfldfrkqnyiinpiniqiqsilvypiflkrgfldekptmidrekflcenkdteladwfmhyknykednyqkfyayplekveekekflunkqinkqkkndvytlmmveyiiqkifgdkfveenplvlkgifqskaerqqnnthaattqernlngilnqpkdikiqgkitvkgvklkdignfrkyeidqrvntfldyeprkewmaylpndwkekekqgqlppnnvidrqiskyetvrskillkdvqelekiisdeikeehrhdlkqgkyynfkyyilngllrqlknenvenykvfklntnpekvnitqlkqeatdleqkafvltyirnkfahnqlpkkefwdycqekygkiekektyaeyfaevfkrekealik Porphyromonas 14mteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsflcalwknfgulae (SEQ IDdndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekeelqanalsldnlksilfdNO: 47)flqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkidhehndevdphyhfnhlvrkgkkdryghndnpsflchhfvdgegmvteagllffvslflekrdaiwmqkkirgflcggtetyqqmtnevfcrsrislpklkleslrmddwmlldmlnelvrcpkplydrlreddracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldyfetgdkpyisqtsphyhiekgkiglrfmpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaervqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmiailsqehkdmeekirkklqemmadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdasgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrvenrpflllkepktdrqtivagwkgefhlprgifteavrdcliemghdevasykevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskeeraeewergkerfrdleawsysaarriedafagieyaspgnkkkieqllrdlslweafesklkvradrinlaklkkeileaqehpyhdfkswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirpnvqeqgslnylnrvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglameqypisklrveyelakyqtarvcvfeltlrleeslltryphlpdesfremleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa Prevotella 15mnipalvenqkkyfgtysvmamlnaqtvldhiqkvadiegeqnennenlwfhpvmshlyna sp. P5-125(SEQ ID kngydkqpektmfiierlqsyfpflkimaenqreysngkykqnrvevnsndifevlkrafgvlkNO: 48)myrdltnhyktyeeklndgcefltsteqplsgminnyytvalrnmnerygyktedlafiqdkrfldvkdaygkkksqvntgfflslqdyngdtqkklhlsgvgialliclfldkqyiniflsrlpifssynaqseerriiirsfginsiklpkdrihseksnksvamdmlnevkrcpdelfttlsaekqsrfriisddhnevlmkrssdrfvplllqyidygklfdhirfhvnmgklryllkadktcidgqtrvrvieqpingfgrleeaetmrkqengtfgnsgirirdfenmkrddanpanypyivdtythyilennkvemfindkedsapllpvieddryvvktipscrmstleipamafhmflfgskkteklivdvhnrykrlfqamqkeevtaeniasfgiaesdlpqkildlisgnahgkdvdafirltvddmltdterrikrflcddrksirsadnkmgkrgfkqistgkladflakdivlfqpsyndgenkitglnyrimqsaiavydsgddyeakqqfklmfekarligkgttephpflykvfarsipanavefyerylierkfyltglsneikkgnrvdvpfirrdqnkwktpamktlgriysedlpvelprqmfdneikshlkslpqmegidfnnanytyliaeymkrvldddfqtfyqwnrnyrymdmlkgeydrkgslqhcftsveereglwkerasrteryrkqasnkirsnrqmrnasseeietildkrlsnsrneyqksekvirryrvqdallfllakktlteladfdgerfklkeimpdaekgilseimpmsftfekggkkytitsegmklknygdffvlasdkrignllelvgsdivskedimeefnkydqcrpeissivfnlekwafdtypelsarvdreekvdflcsilkillnnkninkeqsdilrkirnafdhnnypdkgvveikalpeiamsikkafgeyaimk Flavobacterium 16menlnkildkeneiciskifntkgiaapitekaldnikskqkndlnkearlhyfsighsflqidtkkbranchiophilum (SEQ IDvfdyvlieelkdekplkfitlqkdfftkefsiklqklinsirninnhyvhnfndinlnkidsnvfhflkNO: 49)esfelaiiekyykynkkypldneivlflkelfikdentallnyftnlskdeaieyiltftitenkiwninnehnilniekgkyltfeamlflitiflykneanhllpklydfknnkskqelftffskkftsqdidaeeghlikfrdmiqylnhyptawnndlklesenknkimttklidsiiefelnsnypsfatdiqfkkeakaflfasnkkrnqtsfsnksyneeirhnphikqyrdeiasaltpisfnvkedkfkifvkkhvleeyfpnsigyekfleyndftekekedfglklysnpktnklieridnhklykshgrnqdrfmdfsmrflaennyfgkdaffkcykfydtqeqdeflqsnennddvkfhkgkvttyikyeehlknysywdcpfveennsmsvkisigseekilkiqrnlmiyflenalynenvenqgyklynnyyrelkkdveesiasldliksnpdfkskykkilpkrilhnyapakqdkapenafetilkkadfreeqykklikkaeheknkedfvkrnkgkqfklhfirkacqmmyfkekyntikegnaafekkdpviekrknkehefghhknlnitreefndyckwmfafngndsykkylrdlfsekhffdnqeyknlfessvnleafyaktkelfkkwietnkptnnenrytlenyknlilqkqvfinvyhfskylidknlinsennviqykslenveylisdfyfqsklsidqyktcgklfnklksnkledcllyeiaynyidkknvhkidiqkiltskiiltindantpykisvpfnklerytemiaiknqnnlkarflidlplylsknkikkgkdsagyeiiikndleiedintinnkiindsvkftevlmelekyfilkdkcilsknyidnseipslkqfskywikeneneiinyrniachfhlpfletfdnifinveqkfikeelqnvstindlskpqeylillfikfichnnfylnlfnknesktikndkevkknrylqkfinqvilkkk Myroides 17mkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdngnlknyiiodoratimimus (SEQ IDhvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltisqrirqfremlislvtavdqlrnfythNO: 50)yhhsdivienkvldfinssfvstalhvkdkylktdktkeflketiaaeldilieaykkkqiekkntrfkankredilnaiyneafwsfindkdkdkdketvvakgadayfeknhhksndpdfalnisekgivyllsifitnkemdslkanitgfkgkvdresgnsikymatqriysfhtyrglkqkirtseegvketilmqmidelskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvthpvirkryedrfnyfairfldeffdfpflrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakasyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnyksekplvftgqpiaylsmndihsmlfsiltdnaelkktpeeveaklidqigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmfkeskskwkgyqhielqklfayfdtsksdlelilsnmvmvkdypielidlvicksrtivdfinkylearleyienvitryknsigtpqflavrkecftflkksnytvvsldkqverilsmplfiergfmddkptmlegksykqhkekfadwfvhykensnyqnfydtevyeittedkrekakvtkkikqqqkndvifimmvnymleevlklssndrislnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlcdglvhidnvklkdignfrkyendsrvkefityqsdivwsaylsnevdsnklyvierqldnyesirskellkevqeiecsvynqvankeslkqsgnenfkqyvlqgllpigmdvremlilstdvkfkkeeiiqlgqageveqdlysliyirnkfahnqlpikeffdfcennyrsisdneyyaeyymeifrsikekyan Flavobacterium 18mssknesynkqktfnhykqedkyffggfinnaddnlrqvgkeflarinfnhnnnelasvfkdyfcolumnare (SEQ IDnkeksvakrehalnllsnyfpvleriqkhtnhnfeqtreifellldtikklrdyythhyhkpitinpkiNO: 51)ydflddtlldvlitikkkkvkndtsrellkeklrpeltqlknqkreelikkgkklleenlenavfnhclipfleenktddkqnktvslrkyrkskpneetsititqsglvflmsfflhrkefqvftsglerfkakvntikeeeislnknnivymithwsysyynfkglkhriktdqgvstleqnntthsltntntkealltqivdylskvpneiyetlsekqqkefeedineymrenpenedstfssivshkvirkryenkfnyfamrfideyaelptlrfmvnfgdyikdrqkkilesiqfdseriikkeihlfeklslvteykknvylketsnidlsrfplfpnpsyvmannnipfyidsrsnnldeylnqkkkaqsqnkkrnitfekynkeqskdaiiamlqkeigvkdlqqrstigllscnelpsmlyevivkdikgaelenkiaqkireqyqsirdftldspqkdnipttliktintdssvtfenqpidipriknalqkeltitqeklinvkeheievdnynrnkntykfknqpknkvddkklqrkyvfyrneirqeanwlasdlihfmknkslwkgymhnelqsflaffedkkndcialletvfnlkedciltkglknifikhgnfidfykeylklkedflstestflengfiglppkilkkelskrlkyifivfqkrqfiikeleekknnlyadainlsrgifdekptmipfkkpnpdefaswfvasyqynnyqsfyeltpdiverdkkkkyknlrainkvkiqdyylklmvdtlyqdlfnqpldkslsdfyvskaerekikadakayqklndsslwnkvihlslqnnritanpklkdigkykralqdekiatlltydartwtyalqkpekenendykelhytalnmelqeyekvrskellkqvqelekkildkfydfsnnashpedleiedkkgkrhpnfklyitkallkneseiinlenidieillkyydynteelkekiknmdedekakiintkenynkitnvlikkalvliiirnkmahnqyppkfiydlandvpkkeeeyfatyfnryfetitkelwenkekkdktqv Porphyromonas 19mtegnekpyngtyytledkhfwaaflnlarhnayitlahidrqlayskaditndedilffkgqwkngingivalis (SEQ IDldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkelskkekeelqanalsldnlksilfNO: 52)dflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlvrkgkkdkygnndnpfflchhfvdregtvteagllffvslflekrdaiwmqkkirgfkggteayqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldyfetgdkpyitqttphyhiekgkiglrfvpegqhlwpspevgatrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmiailsqehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgvvadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylearkaflqsigrsdrvenhrifilkepktdrqtivagwkgefhlprgifteavrdcliemgydevgsykevgfmakavplyferaskdryqpfydypfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyhdfkswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirtdvqeqgslnvinrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalameqypisklryeyelakyqtarvcafeqtleleeslltryphlpdknfrkmleswsdplldkwpdlhgnvrlliavrnafshnqypmydetlfssirkydpsspdaieermglniahrlseevkqakemveriiqa Porphyromonas20 mteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsflcalwknfsp. (SEQ IDdndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekeelqanalsldnlksilfdCOT-052 NO: 53)flqklkdfrnyyshyrhsesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlv OH4946rkgkkdryghndnpsflchhfvdsegmvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlreddracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyisqttphyhiekgkiglrfvpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldacladkgirrghlpkqmigilsgerkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrvencpflllkepktdrqtivagwkgefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedryqpfydspfnvgnslkpkkgrflskedraeewergkerfrdleawshsaarrikdafagieyaspgnkkkieqllrdlslweafesklkvradkinlaklkkeileaqehpyhdflcswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirpnvqeqgslnylnrvkpmflpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglameqypisklrveyelakyqtarvcvfeltlrleesllsryphlpdesfremleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa Prevotella 21meddkktkestnmldnkhfwaaflnlarhnvyitvnhinkvlelknkkdqdiiidndqdilaiktintermedia (SEQ IDhwekvngdlnkterlrelmtkhfpfletaiytknkedkeevkqekqakaqsfdslkhclflfleklqNO: 54)earnyyshykysestkepmlekellkkmynifddniqlvikdyqhnkdinpdedflchldrteeefnyyfttnkkgnitasgllffvslflekkdaiwmqqklrgfkdnreskkkmthevfcrsrmllpklrlestqtqdwilldmlnelircpkslyerlqgeyrkkfnvpfdsadedydaeqepfkntivrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkliggqkedrhlthklygferiqefakqnrtdewkaivkdfdtyetseepyisetaphyhlenqkigirfrndndeiwpslktngennekrkykldkqyqaeaflsvhellpmmfyylllkkeepnndkknasivegfikreirdiyklydafangeinniddlekycedkgipkrhlpkqmvailydehkdmaeeakrkqkemvkdtkkllatlekqtqgeiedggrnirllksgeiarwlvndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnlinssnphpflkwtkweecnnilsfyrsyltkkieflnklkpedweknqyflklkepktnretivqgwkngfnlprgiftepirewflothqndseeyekvetldrvglvtkviplfflckedskdkeeylkkdaqkeinncvqpfygfpynvgnihkpdekdflpseerkklwgdkkykfkgykakvkskkltdkekeeyrsylefqswnkferelrlyrnqdivtwllctelidklkveglnveelkklrlkdidtdtakqeknnilnrympmqlpvtvyeiddshnivkdrplhtvyieetktkllkqgnflcalvkdrringlfsfvdtssetelksnpiskslveyelgeyqnarietikdmllleetliekyktlptdnfsdmlngwlegkdeadkarfqndvkllvavrnafshnqypmrnriafaninpfslssadtseekkldianqlkdkthkiikriieiekpietke PIN17_ AFJ07523mkmeddkktkestnmldnkhfwaaflnlarhnvyitvnhinkvlelknkkdqdiiidndqdila 0200(SEQ IDikthwekvngdlnkterlrelmtkhfpfletaiytknkedkeevkqekqakaqsfdslkhclflfle[Prevotella NO: 55)klqearnyyshykysestkepmlekellkkmynifddniqlvikdyqhnkdinpdedflchldrtintermediaeeefnyyfttnkkgnitasgllffvslflekkdaiwmqqklrgflcdnreskkkmthevfcrsrmll 17]pklrlestqtqdwilldmlnelircpkslyerlqgeyrkkfnvpfdsadedydaeqepflcntivrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkliggqkedrhlthklygferiqefakqnrtdewkaivkdfdtyetseepyisetaphyhlenqkigirfrndndeiwpslktngennekrkykldkqyqaeaflsvhellpmmfyylllkkeepnndkknasivegfikreirdiyklydafangeinniddlekycedkgipkrhlpkqmvailydehkdmaeeakrkqkemvkdtkkllatlekqtqgeiedggrnirllksgeiarwlyndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnlinssnphpflkwtkweecnnilsfyrsyltkkieflnklkpedweknqyflklkepktnretlvqgwkngfnlprgiftepirewflothqndseeyekvetldrvglvtkviplffkkedskdkeeylkkdaqkeinncvqpfygfpynvgnihkpdekdflpseerkklwgdkkykfkgykakvkskkltdkekeeyrsylefqswnkferelrlyrnqdivtwllctelidklkveglnveelkklrlkdidtdtakqeknnilnrvmpmqlpvtvyeiddshnivkdrplhtvyieetktkllkqgnflcalvkdrringlfsfvdtssetelksnpiskslveyelgeyqnarietikdmilleetliekyktlptdnfsdmlngwlegkdeadkarfqndvkllvavrnafshnqypmrnriafaninpfslssadtseekkldianqlkdkthkiikriieiekpietke Prevotella BAU18623meddkkttdsisyelkdkhfwaaflnlarhnvyitvnhinkvlelknkkdqdiiidndqdilaiktintermedia (SEQ IDhwekvngdlnkterlrelmtkhfpfletaiysknkedkeevkqekqakaqsfdslkhclflfleklNO: 56) qetrnyyshykysestkepmlekellkkmynifddniqlvikdyqhnkdinpdedfkhldrteedfnyyftrnkkgnitesgllffvslflekkdaiwmqqklrgfkdnreskkkmthevfcrsrmllpklrlestqtqdwilldmlnelircpkslyerlqgedrekfkvpfdpadedydaeqepfkntivrhqdrfpyfalryfdyneiftnlrfqidlgtfhfsiykkliggqkedrhlthklygferiqefakqnrpdewkaivkdldtyetsneryisettphyhlenqkigirfrndndeiwpslktngennekskykldkqyqaeaflsvhellpmmfyylllkkeepnndkknasivegfikreirdmyklydafangeinniddlekycedkgipkrhlpkqmvailydehkdmvkeakrkqrkmvkdtekllaalekqtqektedggrnirllksgeiarwlyndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnlinssnphpflkwtkweecnnilsfyrsyltkkieflnklkpedweknqyflklkepktnretivqgwkngfnlprgiftepirewflothqndskeyekvealdrvglvtkviplffkkedskdkeedlkkdaqkeinncvqpfysfpynvgnihkpdekdflhreerielwdkkkdkfkgykakvkskkltdkekeeyrsylefqswnkferelrlyrnqdivtwllctelidklkveglnveelkklrlkdidtdtakqeknnilnrympmqlpvtvyeiddshnivkdrplhtvyieetktkllkqgnflcalvkdrringlfsfydtsseaelksnpiskslveyelgeygnarletikdmilleetliekyknlptdnfsdmlngwlegkdeadkarfqndvkllvavrnafshnqypmrnriafaninpfslssadtseekkldianqlkdkthkiikriieiekpietke HMPREF6485_ EFU31981mqkqdklfvdrkknaifafpkyitimenkekpepiyyeltdkhfwaaflnlarhnvyttinhinrr 0083(SEQ ID leiaelkddgymmgikgswneqakkldkkvrlrdlimkhfpfleaaayemtnskspnnkeqre[Prevotella NO: 57)keqsealslnnlknvlfifleklqvlrnyyshykyseespkpifetsllknmykvfdanvrlvkrdybuccae mhhenidmqrdfthlnrkkqvgrtkniidspnfhyhfadkegnmtiagllffvslfldkkdaiwATCC mqkklkgflcdgrnlreqmtnevfcrsrislpklklenvqtkdwmqldmlnelvrcpkslyerlre33574]kdresfkvpfdifsddynaeeepfkntivrhqdrfpyfvlryfdlneifeqlrfqidlgtyhfsiynkrigdedevrhlthhlygfariqdfapqnqpeewrklvkdldhfetsqepyisktaphyhlenekigikfcsahnnlfpslqtdktcngrskfnlgtqftaeaflsvhellpmmfyyllltkdysrkesadkvegiirkeisniyaiydafanneinsiadltrrlqntnilqghlpkqmisilkgrqkdmgkeaerkigemiddtqrrldllckqtnqkirigkrnagllksgkiadwlvndmmrfqpvqkdqnnipinnskansteyrmlqralalfgsenfrlkayfnqmnlvgndnphpflaetqwehqtnilsfyrnylearkkylkglkpqnwkqyqhflilkvqktnrntivtgwknsfnlprgiftqpirewfekhnnskriydqilsfdrygfvakaiplyfaeeykdnvqpfydypfnignrlkpkkrqfldkkervelwqknkelfknypsekkktdlayldflswkkferelrliknqdivtwlmflcelfnmatveglkigeihlrdidtntaneesnnilnrimpmklpvktyetdnkgnilkerplatfyieetetkvlkqgnfkalvkdrringlfsfaettdlnleehpiskisvdlelikyqttrisifemtlglekklidkystlptdsfrnmlerwlqckanrpelknyvnsliavrnafshnqypmydatlfaevkkftlfpsvdtkkielniapqlleivgkaikeieksenknHMPREF9144_ EGQ18444mkeeekgktpvvstynkddkhfwaaflnlarhnvyitvnhinkilgegeinrdgyentlekswn 1146(SEQ IDeikdinkkdrlskliikhfpflevttyqrnsadttkqkeekqaeaqsleslkksffvfiyklrdlrnhys[Prevotella NO: 58)hykhskslerpkfeedlqekmynifdasiqlvkedykhntdikteedflchldrkgqfkysfadne ignitesgllffvslflekkdaiwvqkklegflccsnesyqkmtnevfcrsrmllpklrlqstqtqdwilATCCldmlnelircpkslyerlreedrkkfrvpieiadedydaeqepfknalvrhqdrfpyfalryfdynei700821]ftnlrfqidlgtyhfsiykkqigdykeshhlthklygferiqeftkqnrpdewrkfvktfnsfetskepyipettphyhlenqkigirfrndndkiwpslktnseknekskykldksfqaeaflsvhellpmmfyylllktentdndneietkkkenkndkqekhkieeiienkiteiyalydafangkinsidkleeyckgkdieighlpkqmiailksehkdmateakrkqeemladvqkslesldnqineeienverknsslksgeiaswlyndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnliessnphpflnntewekcnnilsfyrsyleakknfleslkpedweknqyflmlkepktncetivqgwkngfnlprgiftepirkwfmehrknitvaelkrvglvakviplffseeykdsvqpfynylfnvgninkpdeknflnceerrellrkkkdefkkmtdkekeenpsylefqswnkferelrlvrnqdivtwllcmelfnkkkikelnvekiylknintnttkkeknteekngeekiikeknnilnrimpmrlpikvygrenfsknkkkkirrntfftvyieekgtkllkqgnfkalerdrrlgglfsfvkthskaesksntisksrveyelgeyqkarieiikdmlaleetlidkynsldtdnfhnmltgwlklkdepdkasfqndvdlliavrnafshnqypmrnriafaninpfslssantseekglgianqlkdkthktiekiieiekpietkeHMPREF9714_ EH008761mkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdngnlknyii02132 (SEQ IDhvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltlsqrirqfremlislvtavdqlrnfyth[Myroides NO: 59)yhhseivienkvldflnsslvstalhvkdkylktdktkeflketiaaeldilleaykkkqiekkntrflcodoratimimusankredilnaiyneafwsfindkdkdketvvakgadayfeknhhksndpdfalnisekgivylls CCUGffltnkemdslkanitgfkgkvdresgnsikymatqriysfhtyrglkqkirtseegvketllmqmi12901]delskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvihpvirkryedrfnyfairfldeffdfptlrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakanyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnvksekplvftgqpiaylsmndihsmlfslltdnaelkktpeeveaklidqigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmteefkskwkgyqhtelqklfayydtsksdldlilsdmvmvkdypielialvkksrtivdflnkylearlgymenvitrvknsigtpqflavrkecftflkksnytvvsldkqverilsmplfiergfmddkptmlegksyqqhkekfadwfvhykensnyqnfydtevyeittedkrekakvtkkikqqqkndvftlmmvnymleevlklssndrlslnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlceglvridkvklkdignfrkyendsrvkefltyqsdivwsaylsnevdsnklyvierqldnyesirskellkevqeiecsvynqvankeslkqsgnenfkqyvlqglvpigmdvremlilstdvkfikeeiiqlgqageveqdlysliyirnkfahnqlpikeffdfcennyrsisdneyyaeyymeifrsikekyts HMPREF9711_ EKB06014mkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdngnlknyii00870 (SEQ IDhvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltlsqrirqfremlislvtavdqlrnfyth[Myroides NO: 60)yhhseivienkvldflnsslvstalhvkdkylktdktkeflketiaaeldilleaykkkqiekkntrfkodoratimimusankredilnaiyneafwsfindkdkdketvvakgadayfeknhhksndpdfalnisekgivylls CCUGffltnkemdslkanitgfkgkvdresgnsikymatqriysfhtyrglkqkirtseegvketllmqmi3837] delskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvihpvirkryedrfnyfairfldeffdfptlrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakasyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnyksekplvftgqpiaylsmndihsmlfslltdnaelkktpeeveaklidqigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmfkeskskwkgyqhtelqklfayfdtsksdlelilsdmvmvkdypielidlyrksrtivdflnkylearlgyienvitrvknsigtpqfktvrkecfaflkesnytvasldkqierilsmplfiergfmdskptmlegksyqqhkedfadwfvhykensnyqnfydtevyeiitedkreqakvtkkikqqqkndvftlmmvnymleevlklpsndrlslnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlceglvridkvklkdignfrkyendsrvkefltyqsdivwsgylsnevdsnklyvierqldnyesirskellkevqeiecivynqvankeslkqsgnenfkqyvlqgllprgtdvremlilstdvkfkkeeimqlgqvreveqdlysliyirnkfahnqlpikeffdfcennyrpisdneyyaeyymeifrsikekyas HMPREF9699_ EKB54193menktslgnniyynpfkpqdksyfagyfnaamentdsvfrelgkrlkgkeytsenffdaifkeni 02005(SEQ IDslveyeryvkllsdyfpmarlldkkevpikerkenfkknflcgiikavrdlrnfythkehgeveitde[Bergeyella NO: 61)ifgvldemlkstvltykkkkvktdktkeilkksiekqldilcqkkleylrdtarkieekrrnqrergezoohelcumkelvapfkysdkrddliaaiyndafdvyidkkkdslkesskakyntksdpqqeegdlkipiskng ATCCvvfllslfltkqeihafkskiagfkatvideatvseatvshgknsicfmatheifshlaykklkrkvrta43767] einygeaenaeqlsvyaketlmmqmldelskvpdvvygnisedvqktfiedwneylkenngdvgtmeeeqvihpvirkryedkfnyfairfldefaqfptlrfqvhlgnylhdsrpkenlisdrrikekitvfgrlselehkkalfikntetnedrehyweifpnpnydfpkenisvndkdfpiagsildrekqpvagkigikvkllnqqyvsevdkavkahqlkqrkaskpsiqniieeivpinesnpkeaivfggqptaylsmndihsilyeffdkwekkkeklekkgekelrkeigkelekkivgkiqaqiqqiidkdtnakilkpyqdgnstaidkeklikdlkqeqnilqklkdeqtvrekeyndfiayqdknreinkvrdrnhkqylkdnlkrkypeaparkevlyyrekgkvavwlandikrfmptdfknewkgeqhsllqkslayyeqckeelknllpekvfqhlpfklggyfqqkylyqfytcyldkrleyisglvqqaenfksenkvfkkvenecfkflkkqnythkeldarvqsilgypiflergfmdekptiikgktfkgnealfadwfryykeyqnfqtfydtenyplvelekkqadrkrktkiyqqkkndvifilmakhifksvfkqdsidqfsledlyqsreerlgnqerarqtgerntnyiwnktvdlklcdgkitvenvklknvgdfikyeydqrvqaflkyeeniewqaflikeskeeenypyvvereieqyekvrreellkevhlieeyilekvkdkeilkkgdnqnfkyyilngllkqlknedvesykvfnlntepedvninqlkqeatdleqkafvltyirnkfahnqlpkkefwdycqekygkiekektyaeyfaevflckekealik HMPREF9151_ EKY00089mmekenvqgshiyyeptdkcfwaafynlarhnayltiahinsfvnskkginnddkvldiiddw 01387(SEQ IDskfdndllmgarinklilkhfpflkaplyqlakrktrkqqgkeqqdyekkgdedpeviqeaianaf[Prevotella NO: 62)kmanvrktlhaflkqledlrnhfshynynspakkmevkfddgfcnklyyvfdaalqmvkddnr imnpeinmqtdfehlvrlgrnrkipntfkynftnsdgtinnngllffvslflekrdaiwmqkkikgfF0055] kggtenymrmtnevfcrnrmvipklrletdydnhqlmfdmlnelvrcplslykrlkqedqdkfrvpiefldedneadnpygenansdenpteetdplkntivrhqhrfpyfvlryfdlnevflcqlrfqinlgcyhfsiydktigertekrhltrtlfgfdrlqnfsvklqpehwknmvkhldteessdkpylsdamphyqienekigihflktdtekketvwpsleveevssnrnkykseknitadaflsthellpmmfyyqllsseektraaagdkvqgvlqsyrkkifdiyddfangtinsmqklderlakdnllrgnmpqqmlailehqepdmeqkakekldrlitetkkrigkledqflcqkvrigkrradlpkvgsiadwlvndmmrfqpakrnadntgvpdskansteyrllqealafysaykdrlepyfrqvnliggtnphpflhrvdwkkenhllsfyhdyleakeqylshlspadwqkhqhflllkvrkdiqnekkdwkkslvagwkngfnlprglftesiktwfstdadkvqitdtklfenrvgliakliplyydkvyndkpqpfyqypfnindrykpedtrkrftaassklwnekkmlyknaqpdssdkieypqyldflswkklerelrmlrnqdmmvwlmckdlfaqctvegvefadlklsqlevdvnvqdnlnylnnvssmilplsvypsdaqgnvlrnskplhtvyvqenntkllkqgnflcsllkdrringlfsfiaaegedlqqhpltknrleyelsiyqtmrisvfeqtlqlekailtrnktlcgnnfnnllnswsehrtdkktlqpdidfliavrnafshnqypmstntvmqgiekfniqtpkleekdglgiasqlakktkdaasrlqniinggtn A343_1752 EOA10535mteqnekpyngtyytledkhfwaaffnlarhnayitlthidrqlayskaditndedilffkgqwkn[Porphyromonas (SEQ IDldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekeelqanalsldnlksilfgingivalis NO: 63)dflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhl JCVIvrkgkkdregnndnpfflchhfvdreekvteagllffvslflekrdaiwmqldcirgfkggtetyqqSC001]mtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldyfetgdkpyitqttphyhiekgkiglrfvpegqllwpspevgatrtgrskyaqdkrftaeaflsvhelmpmmfyyfllrekyseeasaervqgrikrviedvyavydafargeidtldrldacladkgirrghlprqmiailsqehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdrvenhrflllkepktdrqtivagwkgefhlprgifteavrdcliemgldevgsykevgfmakavplyferackdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrdleawshsaarriedafagienasrenkkkieqllqdlslwetfesklkvkadkiniaklkkeileakehpyldfkswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirtdvheqgslnylnrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnflcsfvkdrringlfsfvdtgalameqypisklrveyelakyqtarvcafeqtleleeslltryphlpdknfrkmleswsdplldkwpdlhgnvrlliavrnafshnqypmydetlfssirkydpsspdaieermglniahrlseevkqakemveriiqa HMPREF1981_ ERI81700mesiknsqkstgktlqkdppyfglylnmallnyrkvenhirkwlgdvallpeksgfhsllttdnlss03090 (SEQ IDakwtrfyyksrkflpflemfdsdkksyenrrettecldtidrqkissllkevygklqdirnafshyhi[Bacteroides NO: 64)ddqsvkhtaliissemhrfienaysfalqktrarftgvfvetdflqaeekgdnkkffaiggnegiklkpyogenesdnalifliclfldreeafkflsratgflcstkekgflavretfcalccrqpherllsvnpreallmdmlnelF0041]nrcpdilfemldekdqksflpllgeeeqahilenslndelceaiddpfemiaslskrvryknrfpylmlryieeknllpfirfridlgclelasypkkmgeennyersvtdhamafgrltdfhnedavlqqitkgitdevrfslyapryaiynnkigfvrtggsdkisfptlkkkggeghcvaytlqntksfgfisiydlrkilllsfldkdkaknivsglleqcekhwkdlsenlfdairtelqkefpvplirytlprskggklvsskladkqekyeseferrkeklteilsekdfdlsqiprrmidewlnvlptsrekklkgyvetlkldcrerlrvfekrekgehpvpprigematdlakdiirmvidqgvkqritsayyseiqrclaqyagddnrrhldsiirelrlkdtknghpflgkvlrpglghteklyqryfeekkewleatfypaaspkrvprfvnpptgkqkelpliirnlmkerpewrdwkqrknshpidlpsqlfeneicrllkdkigkepsgklkwnemfklywdkefpngmqrfyrckrrvevfdkvveyeyseeggnykkyyealidevvrqkissskeksklqvedltlsvrrvflcrainekeyqlrllceddrllfmavrdlydwkeaqldldkidnmlgepvsysqviqleggqpdavikaecklkdvsklmrycydgrvkglmpyfanheatqeqvemelrhyedhrrrvfnwvfaleksvlknekliffyeesqggcehrrcidalrkaslvseeeyeflvhirnksahnqfpdleigklppnvtsgfceciwskykaiicriipfidperrffgklleqk HMPREF1553_ ERJ65637mntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllcdhllsv02065 (SEQ IDdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslldflrndfshnrldgttf[Porphyromonas NO: 65)ehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrkeqlisvadgkecltvsglafficlflgingivalisdreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydF0568]mlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnprsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmdqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlqkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrhqfraivaelrlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkykdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpi ndlHMPREF1988_ ERJ81987mntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllcdhllsv01768 (SEQ IDdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslldflrndfshnrldgttf[Porphyromonas NO: 66)ehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrkeqlisvadgkecltvsglafficlflgingivalisdreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydF0185]mlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsgflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkykdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildhenrffgkllnnmsqpi ndlHMPREF1 ERJ87335mntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllcdhllsv990_01800 (SEQ IDdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslldflrndfshnrldgttf[Porphyromonas NO: 67)ehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrkeqlisvadgkecltvsglafficlflgingivalisdreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydW4087]mlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnprsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmdqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlqkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrhqfraivaelrlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskvmellkykdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvrdkkrelrtagkpvppdlaayikrsfhravnerefmlrlvqeddrlmlmainkimtdreedilpglknidsildkenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaeipliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpindlM573_117042 KJJ86756mkmeddkkttestnmldnkhfwaaflnlarhnvyitvnhinkvlelknkkdqdiiidndqdilai[Prevotella (SEQ IDkthwekvngdlnkterlrelmtkhfpfletaiytknkedkeevkqekqaeaqsleslkdclflflekintermedia NO: 68)lqearnyyshykysestkepmleegllekmynifddniqlvikdyqhnkdinpdedflchldrkg ZT]qfkysfadnegnitesgllffvslflekkdaiwmqqkltgfkdnreskkkmthevfcrrrmllpklrlestqtqdwilldmlnelircpkslyerlqgeyrkkfnvpfdsadedydaeqepfkntivrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkliggqkedrhlthklygferiqefakqnrpdewkalvkdldtyetsneryisettphyhlenqkigirfrngnkeiwpslktngennekskykldkpyqaeaflsvhellpmmfyylllkkeepnndkknasivegfikreirdmyklydafangeinnigdlekycedkgipkrhlpkqmvailydepkdmvkeakrkqkemvkdtkkllatlekqtqeeiedggrnirllksgeiarwlyndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnlinssnphpflkwtkweecnnilsfyrnyltkkieflnklkpedweknqyflklkepktnretivqgwkngfnlprgiftepirewflcrhqndskeyekvealkrvglvtkviplffkeeyfkedaqkeinncvqpfysfpynvgnihkpdekdflpseerkklwgdkkdkfkgykakvkskkltdkekeeyrsylefqswnkferelrlyrnqdivtwllctelidkmkveglnveelqklrlkdidtdtakqeknnilnrimpmqlpvtvyeiddshnivkdrplhtvyieetktkllkqgnfkalvkdrringlfsfvdtsskaelkdkpisksvveyelgeyqnarietikdmillektlikkyeklptdnfsdmlngwlegkdesdkarfqndvkllvavrnafshnqypmrnriafaninpfslssadiseekkldianqlkdkthkiikkiieiekpietke A2033_10205 OFX18020.1menqtqkgkgiyyyytknedkhyfgsflnlannnieqiieefrirlslkdeknikeiinnyftdkks[Bacteroidetes (SEQ IDytdwerginilkeylpvidyldlaitdkefekidlkqketakrkyfrtnfsllidtiidlrnfythyfhkbacterium NO: 69)pisinpdvakfldknllnycldikkqkmktdktkqalkdgldkelkklielkkaelkekkiktwniGWA2_31_9tenvegavyndafnhmvyknnagvtilkdyhksilpddkidselklnfsisglvfllsmflskkeieqfksnlegfkgkvigengeyeiskfnnslkymathwifsyltflcglkqrvkntfdketllmqmidelnkvphevyqtlskeqqnefledineyvqdneenkksmensivvhpvirkryddkfnyfairfldefanfptlkffvtagnfvhdkrekqiqgsmltsdrmikekinvfgklteiakyksdyfsnentletsewelfpnpsylliqnnipvhidlihnteeakqcqiaidrikettnpakkrntrkskeeiikiiyqknknikygdptallssnelpaliyellvnkksgkeleniivekivnqyktiagfekgqnlsnslitkklkksepnedkinaekiilainreleitenklniiknnraefrtgakrkhifyskelgqeatwiaydlkrfmpeasrkewkgfhhselqkflafydrnkndakallnmfwnfdndqligndlnsafrefhfdkfyekylikrdeilegfksfisnflcdepkllkkgikdiyrvfdkryyiikstnaqkeqllskpiclprgifdnkptyiegvkvesnsalfadwyqytysdkhefqsfydmprdykeqfekfelnniksiqnkknlnksdkfiyfrykqdlkikqiksqdlfiklmvdelfnvvfknnielnlkklyqtsderfknqliadvqknrekgdtsdnkmnenfiwnmtiplslcngqieepkvklkdigkfrkletddkviqlleydkskvwkkleiedelenmpnsyerirrekllkgiqefehfllekekfdginhpkhfeqdlnpnfktyvingvlrknsklnyteidklldlehisikdietsakeihlayflihvrnkfghnqlpkleafelmkkyykknneetyaeyfhkvssqivnefknslekhs SAMN05421542_ SDI27289.1mektqtglgiyydhtklqdkyffggffnlaqnnidnvikafiikffperkdkdiniaqfldicfkdn 0666(SEQ IDdadsdfqkknkflrihfpvigfltsdndkagfkkkfalllktiselrnfythyyhksiefpselfelldd[Chryseo- NO: 70)ifvkttseikklkkkddktqqllnknlseeydiryqqqierlkelkaqgkrvsltdetairngvfnaafbacteriumnhliyrdgenvkpsrlyqssysepdpaengislsqnsilfllsmflerketedlksrvkgflcakiikqjejuense]geeqisglkfmathwvfsylcfkgikqklstefheetlliqiidelskvpdevysafdsktkekfledineymkegnadlsledskvihpvirkryenkfnyfairfldeylsstslkfqvhvgnyvhdrrvkhingtgfqterivkdrikvfgrlsnisnlkadyikeqlelpndsngweifpnpsyifidnnvpihvladeatkkgielfkdkrrkeqpeelqkrkgkiskynivsmiykeakgkdklrideplallslneipallyqilekgatpkdieliiknklterfekiknydpetpapasqiskrlrnnttakgqealnaeklslliereientetklssieekrlkakkeqrrntpqrsifsnsdlgriaawladdikrfmpaeqrknwkgyqhsqlqqslayfekrpqeaflllkegwdtsdgssywnnwymnsflennhfekfyknylmkrvkyfselagnikqhthntkflrkfikqqmpadlfpkrhyilkdleteknkvlskplvfsrglfdnnptfikgvkvtenpelfaewysygyktehvfqhfygwerdynelldselqkgnsfaknsiyynresqldliklkqdlkikkikiqdlflkriaeklfenvfnypttlsldefyltqeeraekerialaqslreegdnspniikddfiwsktiafrskqiyepaiklkdigkfnrfvlddeeskaskllsydknkiwnkeqlerelsigensyevirreklfkeignielqilsnwswdginhprefemedqkntrhpnfkmylvngilrkninlykededfwleslkendflalpsevletksemvqllflvilirnqfahnqlpeiqfynfirknypeiqnntvaelylnliklavqklkdns SAMN05444360_ SHM52812.1mntrvtgmgvsydhtkkedkhffggflnlaqdnitavikafcikfdknpmssvqfaescftdkds 11366(SEQ IDdtdfqnkvryvrthlpvigylnyggdrntfrqklstllkavdslrnfythyyhsplalstelfelldtvf[Chryseo- NO: 71)asvavevkqhkmkddktrqllskslaeeldirykqqlerlkelkeqgknidlrdeagirngvinaabacteriumfnhliykegeiakptlsyssfyygadsaengitisqsgllfllsmflgkkeiedlksrirgfkakivrdgcarnipullorum]eenisglkfmathwifsylsflcgmkqrlstdfheetlliqiidelskvpdevyhdfdtatrekfvedineyiregnedfslgdstiihpvirkryenkfnyfavrfldefikfpslrfqvhlgnfvhdrrikdihgtgfqtervvkdrikvfgklseisslkteyiekeldldsdtgweifpnpsyvfidnnipiyistnktfkngssefiklrrkekpeemkmrgedkkekrdiasmignagslnsktplamlslnempallyeilvkkttpeeieliikekldshfeniknydpekplpasqiskrlrnnttdkgkkvinpeklihlinkeidateakfallaknrkelkekfrgkplrqtifsnmelgreatwladdikrfmpdilrknwkgyqhnqlqqslaffnsrpkeaftilqdgwdfadgssfwngwiinsfvknrsfeyfyeayfegrkeyfsslaenikqhtsnhrnlrrfidqqmpkglfenrhyllenleteknkilskplvfprglfdtkptfikgikvdeqpelfaewyqygystehvfqnfygwerdyndlleselekdndfsknsihysrtsqleliklkqdlkikkikiqdlflkliaghifenifkypasfsldelyltqeerinkeqealiqsqrkegdhsdniikdnfigsktvtyeskqisepnvklkdigkfnrfllddkvktllsynedkvwnkndldlelsigensyevirreklfkkiqnfelqtltdwpwngtdhpeefgttdnkgvnhpnfkmyvvngilrkhtdwfkegednwlenlnethfknlsfqeletksksiqtafliimirnqfahnqlpavqffefiqkkypeiqgsttselylnfinlavvellellek SAMN05421786_ SIS70481.1metqilgngisydhtktedkhffggflntaqnnidllikayiskfessprklnsvqfpdvcflcknds1011119 (SEQ IDdadfqhklqfirkhlpviqylkyggnrevlkekifillqavdslrnfythfyhkpiqlpnelltlldtif[Chryseo- NO: 72)geignevrqnkmkddktrhllkknlseeldfryqeqlerlrklksegkkvdlrdteairngvinaafbacteriumnhliflcdaedflcptvsyssyyydsdtaengisisqsgllfllsmflgrremedlksrvrgfkariikhureilyticum]eeqhvsglkfmathwvfsefcflcgiktrinadyheetlliqlidelskvpdelyrsfdvatrerfiedineyirdgkedkslieskivhpvirkryeskfnyfairfldefvnfptlrfqvhagnyvhdrriksiegtgfkterlykdrikvfgklstisslkaeylakavnitddtgwellphpsyvfidnnipihltvdpsflaigvkeyqekrklqkpeemknrqggdkmhkpaisskigkskdinpespvallsmneipallyeilvkkaspeeveakirqkltavferirdydpkvplpasqvskrlrnntdtlsynkeklvelankeveqterklalitknrrecrekvkgkfkrqkvfknaelgteatwlandikrfmpeeqkknwkgyqhsqlqqslaffesrpgearsllqagwdfsdgssfwngwvmnsfardntfdgfyesylngrmkyflrladniaqqsstnklisnfikqqmpkglfdrrlymledlateknkilskplifprgifddkptfkkgvqvseepeafadwysygydvkhkfqefyawdrdyeellreelekdtaftknsihysresqiellakkqdlkvkkvriqdlylklmaeflfenvfghelalpldqfyltqeerlkqeqeaivqsqrpkgddspnivkenfiwsktipfksgrvfepnvklkdigkfrnlltdekvdillsynnteigkqvieneliigagsyefirreqlfkeiqqmkrlslrsvrgmgvpirinlk Prevotella WP_mqkqdklfvdrkknaifafpkyitimenqekpepiyyeltdkhfwaaflnlarhnvyttinhinrrbuccae 004343581leiaelkddgymmdikgswneqakkldkkvrlrdlimkhfpfleaaayeitnskspnnkeqrek (SEQ IDeqsealslnnlknvlfifleklqvlrnyyshykyseespkpifetsllknmykvfdanvrlvkrdyNO: 73 mhhenidmqrdfthlnrkkqvgrtkniidspnfhyhfadkegnmtiagllffvslfldkkdaiwmqkklkgflcdgrnlreqmtnevfcrsrislpklklenvqtkdwmqldmlnelvrcpkslyerlrekdresfkvpfdifsddydaeeepfkntivrhqdrfpyfvlryfdlneifeqlrfqidlgtyhfsiynkrigdedevrhlthhlygfariqdfaqqnqpevwrklvkdldyfeasqepyipktaphyhlenekigikfcsthnnlfpslktektcngrskfnlgtqftaeaflsvhellpmmfyyllltkdysrkesadkvegiirkeisniyaiydafangeinsiadltcrlqktnilqghlpkqmisilegrqkdmekeaerkigemiddtqrrldllckqtnqkirigkrnagllksgkiadwlvndmmrfqpvqkdqnnipinnskansteyrmlqralalfgsenfrlkayfnqmnlvgndnphpflaetqwehqtnilsfyrnylearkkylkglkpqnwkqyqhflilkvqktnrntivtgwknsfnlprgiftqpirewfekhnnskriydqilsfdrygfvakaiplyfaeeykdnvqpfydypfnignklkpqkgqfldkkervelwqknkelflcnypsekkktdlayldflswkkferelrliknqdivtwlmflcelfnmatveglkigeihlrdidtntaneesnnilnrimpmklpvktyetdnkgnilkerplatfyieetetkvlkqgnfkvlakdrringllsfaettdidleknpitklsvdhelikyqttrisifemtlglekklinkyptlptdsfrnmlerwlqckanrpelknyvnsliavrnafshnqypmydatlfaevkkftlfpsvdtkkielniapqlleivgkaikeieksenk nPorphyromonas WP_mntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllcdhllsvgingivalis 005873511drwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslldflrndfshnrldgttf(SEQ IDehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrkeqlisvadgkecltvsglafficlflNO: 74)dreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhnlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaellilldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpi ndlPorphyromonas WP_mtecinekpyngtyytledkhfwaaffnlarhnayitlahidrqlayskaditndedilfflcgqwkngingivalis 005874195ldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekeelqanalsldnlksilf(SEQ IDdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlNO: 75)vrkgkkdkygnndnpfflchhfvdreekvteagllffvslflekrdaiwmqkkirgfkggteayqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyitqttphyhiekgkiglrfvpegqllwpspevgatrtgrskyaqdkrftaeaflsvhelmpmmfyyfllrekyseeasaekvqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmiailsqehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdreenhrflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdryqpfydypfnvgnslkpkkgrflskekraeewesgkerfrdleawshsaarriedafvgieyaswenkkkieqllqdlslwetfesklkvkadkiniaklkkeileakehpyhdfkswqkferelrlvknqdiitwmmerdlmeenkvegldtgtlylkdirtdvqeqgslnylnhvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalameqypisklrveyelakyqtarvcafeqtleleeslltryphlpdesfremleswsdplldkwpdlqrevrlliavrnafshnqypmydetifssirkydpssldaieermglniahrlseevklakemveriiqa Prevotella WP_mkeeekgktpvvstynkddkhfwaaflnlarhnvyitvnhinkilgegeinrdgyentlekswn pallens006044833eikdinkkdrlskliikhfpflevttyqrnsadttkqkeekqaeaqsleslkksffvfiyklrdlrnhys(SEQ IDhykhskslerpkfeedlqekmynifdasiqlvkedykhntdikteedflchldrkgqfkysfadneNO: 76)gnitesgllffvslflekkdaiwvqkklegflccsnesyqkmtnevfcrsrmllpklrlqstqtqdwilldmlnelircpkslyerlreedrkkfrvpieiadedydaeqepflcnalvrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkqigdykeshhlthklygferiqeftkqnrpdewrkfvktfnsfetskepyipettphyhlenqkigirfrndndkiwpslktnseknekskykldksfqaeaflsvhellpmmfyylllktentdndneietkkkenkndkqekhkieeiienkiteiyalydafangkinsidkleeyckgkdieighlpkqmiailksehkdmateakrkqeemladvqkslesldnqineeienverknsslksgeiaswlyndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnliessnphpflnntewekcnnilsfyrsyleakknfleslkpedweknqyflmlkepktncetivqgwkngfnlprgiftepirkwfmehrknitvaelkrvglvakviplffseeykdsvqpfynylfnvgninkpdeknflnceerrellrkkkdefkkmtdkekeenpsylefqswnkferelrlyrnqdivtwllcmelfnkkkikelnvekiylknintnttkkeknteekngeekiikeknnilnrimpmrlpikvygrenfsknkkkkirrntfftvyieekgtkllkqgnfkalerdrrlgglfsfvkthskaesksntisksrveyelgeyqkarieiikdmlaleetlidkynsldtdnfhnmltgwlklkdepdkasfqndvdlliavrnafshnqypmrnriafaninpfslssantseekglgianqlkdkthktiekiieiekpietke MyroidesWP_ mkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdngnlknyiiodoratimimus 006261414hvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltlsqrirqfremlislvtavdqlrnfyth(SEQ IDyhhseivienkvldflnsslvstalhvkdkylktdktkeflketiaaeldilleaykkkqiekkntrfkNO: 77)ankredilnaiyneafwsfindkdkdketvvakgadayfeknhhksndpdfalnisekgivyllsffltnkemdslkanitgfkgkvdresgnsikymatqriysfhtyrglkqkirtseegvketllmqmidelskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvihpvirkryedrfnyfairfldeffdfptlrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakanyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnvksekplvftgqpiaylsmndihsmlfslltdnaelkktpeeveaklidqigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmteefkskwkgyqhtelqklfayydtsksdldlilsdmvmvkdypielialvkksrtivdflnkylearlgymenvitrvknsigtpqflctvrkecftflkksnytvvsldkqverilsmplfiergfmddkptmlegksyqqhkekfadwfvhykensnyqnfydtevyeittedkrekakvtkkikqqqkndvftlmmvnymleevlklssndrlslnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlceglvridkvklkdignfrkyendsrvkefltyqsdivwsaylsnevdsnklyvierqldnyesirskellkevqeiecsvynqvankeslkqsgnenfkqyvlqglvpigmdvremlilstdvkfikeeiiqlgqageveqdlysliyirnkfahnqlpikeffdfcennyrsisdneyyaeyymeifrsikekyts Myroides WP_mkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdngnlknyiiodoratimimus 006265509hvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltlsqrirqfremlislvtavdqlrnfyth(SEQ IDyhhseivienkvldflnsslvstalhvkdkylktdktkeflketiaaeldilleaykkkqiekkntrfkNO: 78)ankredilnaiyneafwsfindkdkdketvvakgadayfeknhhksndpdfalnisekgivyllsffltnkemdslkanitgfkgkvdresgnsikymatqriysfhtyrglkqkirtseegvketllmqmidelskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvihpvirkryedrfnyfairfldeffdfptlrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakasyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnvksekplvftgqpiaylsmndihsmlfslltdnaelkktpeeveaklidqigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmfkeskskwkgyqhtelqklfayfdtsksdlelilsdmvmvkdypielidlyrksrtivdflnkylearlgyienvitrvknsigtpqfktvrkecfaflkesnytvasldkqierilsmplfiergfmdskptmlegksyqqhkedfadwfvhykensnyqnfydtevyeiitedkreqakvtkkikqqqkndvftlmmvnymleevlklpsndrlslnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlceglvridkvklkdignfrkyendsrvkefltyqsdivwsgylsnevdsnklyvierqldnyesirskellkevqeiecivynqvankeslkqsgnenfkqyvlqgllprgtdvremlilstdvkfkkeeimqlgqvreveqdlysliyirnkfahnqlpikeffdfcennyrpisdneyyaeyymeifrsikekyas Prevotella WP_mqkqdklfvdrkknaifafpkyitimenqekpepiyyeltdkhfwaaflnlarhnvyttinhinrrsp. MSX73 007412163leiaelkddgymmgikgswneqakkldkkvrlrdlimkhfpfleaaayeitnskspnnkeqrek (SEQ IDeqsealslnnlknvlfifleklqvlrnyyshykyseespkpifetsllknmykvfdanvrlvkrdyNO: 79) mhhenidmqrdfthlnrkkqvgrtkniidspnfhyhfadkegnmtiagllffvslfldkkdaiwmqkklkgflcdgrnlreqmtnevfcrsrislpklklenvqtkdwmqldmlnelvrcpkslyerlrekdresfkvpfdifsddydaeeepfkntivrhqdrfpyfvlryfdlneifeqlrfqidlgtyhfsiynkrigdedevrhlthhlygfariqdfapqnqpeewrklvkdldhfetsqepyisktaphyhlenekigikfcsthnnlfpslkrektcngrskfnlgtqftaeaflsvhellpmmfyyllltkdysrkesadkvegiirkeisniyaiydafanneinsiadltcrlqktnilqghlpkqmisilegrqkdmekeaerkigemiddtqrrldllckqtnqkirigkrnagllksgkiadwlvsdmmrfqpvqkdtnnapinnskansteyrmlqhalalfgsessrlkayfrqmnlvgnanphpflaetqwehqtnilsfyrnylearkkylkglkpqnwkqyqhflilkvqktnrntivtgwknsfnlprgiftqpirewfekhnnskriydqilsfdrvgfvakaiplyfaeeykdnvqpfydypfnignklkpqkgqfldkkervelwqknkelfknypseknktdlayldflswkkferelrliknqdivtwlmflcelflattveglkigeihlrdidtntaneesnnilnrimpmklpvktyetdnkgnilkerplatfyieetetkvlkqgnfkvlakdrringllsfaettdidleknpitklsvdyelikyqttrisifemtlglekklidkystlptdsfrnmlerwlqckanrpelknyvnsliavrnafshnqypmydatlfaevkkftlfpsvdtkkielniapqlleivgkaikeieksenknPorphyromonas WP_mtegnerpyngtyytledkhfwaaffnlarhnayitlahidrqlayskaditndedilffkgqwkngingivalis 012458414ldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekeelqanalsldnlksilf(SEQ IDdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlNO: 80)vrkgkkdrygnndnpffkhhfvdreekvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldyfetgdkpyitqttphyhiekgkiglrfvpegqhlwpspevgatrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekysdeasaervqgrikrviedvyavydafargeintrdeldacladkgirrghlprqmigilsgehkdmeekvrkklqemivdtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdrvenhrflllkepktdrqtivagwkgefhlprgifteavrdcliemgldevgsykevgfmakavplyferackdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyldfkswqkferelrlyknqdiitwmicrdlmeenkvegldtgtlylkdirtdvqeqgnlnylnrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalameqypisklrveyelakyqtarvcafeqtleleeslltryphlpdknfrkmleswsdplldkwpdlhgnvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqakemaeriiqa Paludibacter WP_mktsanniyfnginsfkkifdskgaiapiaekscrnfdikaqndynkeqrihyfavghtflcqldtepropionicigenes 013446107nlfeyvldenlrakrptrfislqqfdkefienikrlisdirninshyihrfdplkidavptniidflkesfe(SEQ IDlaviqiylkekginylqfsenphadqklvaflhdkflpldekktsmlqnetpqlkeykeyrkyflalNO: 81)skqaaidqllfaeketdyiwnlfdshpvltisagkylsfysclfllsmflykseamiliskikgflckntteeekskreiftffskrfnsmdidseenqlvkfrdlilylnhypvawnkdleldssnpamtdklkskiieleinrsfplyegnerfatfakyqiwgkkhlgksiekeyinasftdeeitaytyetdtcpelkdahkkladlkaakglfgkrkeknesdikktetsirelqhepnpikdkliqrieknlltvsygrnqdrfmdfsarflaeinyfgqdasfkmyhfyatdeqnselekyelpkdkkkydslkfhqgklvhfisykehlkryeswddafviennaiqlklsfdgventvtiqralliylledalrniqnntaenagkqllqeyyshnkadlsaflqiltqqdsiepqqktefkkllprrllnnyspainhlqtphsslplilekallaekrycslvvkakaegnyddfikrnkgkqfklqfirkawnlmyfrnsylqnvqaaghhksfhierdefndfsrymfafeelsqykyylnemfekkgffennefkilfqsgtslenlyektkqkfeiwlasntaktnkpdnyhlnnyeqqfsnqlffinlshfinylkstgklqtdangqiiyealnnvqylipeyyytdkpersesksgnklynklkatkledallyemamcylkadkqiadkakhpitklltsdvefnitnkegiqlyhllvpflckidafiglkmhkeqqdkkhptsflanivnylelvkndkdirktyeafstnpvkrtltyddlakidghlisksikftnvtleleryfifkeslivkkgnnidfkyikglrnyynnekkknegirnkafhfgipdsksydqlirdaevmfianevkpthatkytdlnkqlhtvcdklmetvhndyfskegdgkkkreaagqkyfeniisak Porphyromonas WP_mteqnekpyngtyytledkhfwaaffnlarhnayitlahidrqlayskaditndedilfflcgqwkngingivalis 013816155ldndlerkarlrslilkhfsflegaaygkklfesqssgnkssknkeltkkekeelqanalsldnlksilf(SEQ IDdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlNO: 82)vrkgkkdrygnndnpffichhfvdregtvteagliffvslflekrdaiwmqkkirgflcggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpvdilsdeedtdgaeedpfkntivrhqdrfpyfalryfdlkkvftslrfqidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrivrdldyfetgdkpyitqttphyhiekgkiglrfvpegqhlwpspevgatrtgrskyaqdkrftaeaftsahelmpmmfyyfilrekyseeasaervqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmigilsqehkdmeekirkklqemmadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdrvenhrfifikepktdrqtivagwkgefhlprgifteavrdcliemgldevgsykevgfmakavplyferackdwvqpfynypfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyldfkswqkferelrivknqdiitwmicgdlmeenkvegldtgtlylkdirtdvqeqgslnvinrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalameqypisklrveyelakyqtarvcafeqtleleeslltrcphlpdknfrkmleswsdpildkwpdlhrkvriliavrnafshnqypmydeavfssirkydpsfpdaieermglniahrlseevkqaketveriiqa FlavobacteriumWP_ mssknesynkqktfnhykqedkyffggfinnaddnlrqvgkefktrinfnhnnnelasvfkdyfcolumnare 014165541nkeksvakrehalnllsnyfpvleriqkhtnhnfeqtreifellldtikklrdyythhyhkpitinpki(SEQ IDydflddtlldvlitikkkkvkndtsrellkeklrpeltqlknqkreelikkgkklleenlenavfnhclrNO: 83)pfleenktddkqnktvslrkyrkskpneetsititqsglvflmsfflhrkefqvftsglegfkakvntikeeeislnknnivymithwsysyynfkglkhriktdqgvstleqnntthsltntntkealltqivdylskvpneiyetlsekqqkefeedineymrenpenedstfssivshkvirkryenkfnyfamrfldeyaelptlrfmvnfgdyikdrqkkilesiqfdseriikkeihlfeklslvteykknvylketsnidlsrfplfpnpsyvmannnipfyidsrsnnldeylnqkkkaqsqnkkrnitfekynkeqskdaiiamlqkeigvkdlqqrstigllscnelpsmlyevivkdikgaelenkiaqkireqyqsirdftldspqkdnipttliktintdssvtfenqpidiprlknaiqkeltitqekllnykeheievdnynrnkntykfknqpknkvddkklqrkyvfyrneirqeanwlasdlihfmknkslwkgymhnelqsflaffedkkndcialletvfnlkedciltkglknlflkhgnfidfykeylklkedfintesiftengliglppkilkkelskrflcyifivfqkrqfiikeleekknnlyadainlsrgifdekptmipfkkpnpdefaswfvasyqynnyqsfyeltpdiverdkkkkyknlrainkvkiqdyylklmvdtlyqdlfnqpldkslsdfyvskaerekikadakayqkrndsslwnkvihlslqnnritanpklkdigkykralqdekiatlltyddrtwtyalqkpekenendykelhytalnmelqeyekvrskellkqvqelekqileeytdflstqihpadferegnpnfkkylahsileneddldklpekveamreldetitnpiikkaivliiirnkmahnqyppkfiydlanrfvpkkeeeyfatyfnrvfetitkelwenkekkdktqv Psychroflexus WP_mesiiglglsfnpyktadkhyfgsfinlvennlnavfaefkerisykakdenissliekhfidnmsitorquis 015024765vdyekkisilngylpiidflddelennintrvknfkknfiilaeaieklrdyythfyhdpitfednkep(SEQ IDllelldevilktildvkkkylktdktkeilkdslreemdllvirktdelrekkktnpkiqhtdssqiknsNO: 84)ifndafqgllyedkgnnkktqvshraktrinpkdihkqeerdfeiplstsglvflmslflskkeiedfksnikgfkgkvvkdenhnslkymathrvysilaflcglkyriktdtfsketimmqmidelskvpdcvygnisetkqkdfiedwneyflcdneentenlensrvvhpvirkryedkfnyfairfldefanflctikfqvfmgyyihdqrtktigttnittertvkekinvfgklskmdnlkkhffsqlsddentdweffpnpsynfltqadnspannipiylelknqqiikekdaikaevnqtqnrnpnkpskrdllnkilktyedfhqgdptailslneipallhlfivkpnnktgqqieniirikiekqfkainhpsknnkgipkslfadtnvrynaiklkkdleaeldmlnkkhiafkenqkassnydkllkehqftpknkrpelrkyvfyksekgeeatwlandikrfmpkdfktkwkgcqhselqrklafydrhtkqdikellsgcefdhslldinayfqkdnfedffskylenrietlegylkklhdfkneptplkgvflmcfldlkrqnyvtespeiikkrilakpfflprgvfderptmkkgknplkdknefaewfveylenkdyqkfynaeeyrmrdadfkknavikkqklkdfytlqmvnyllkevfgkdemnlqlselfqtrqerlklqgiakkqmnketgdssentrnqtyiwnkdvpvsffngkvtidkvklknigkykryerdervktfigyevdekwmmylphnwkdrysvkpinvidlqiqeyeeirshellkeignieqyiydhttdknillqdgnpnfkmyvinglligikqvnipdfivlkqntnfdkidftgiascselekktiiliairnkfahnqlpnkmiydlaneflkieknetyanyylkvlkkmisdla Riemerella WP_mffsfhnaqrvifkhlykafdaslrmvkedykahftvnitrdfahlnrkgknkqdnpdfnryrfeanatipestifer 015345620kdgfftesgllfftnlfldkrdaywmlkkvsgfkashkqrekmttevfcrsrillpklrlesrydhnq(SEQ IDmlldmlselsrcpkllyeklseenkkhfqveadgfldeieeeqnpfkdtlirhqdrfpyfalryldlnNO: 85)esflcsirfqvdlgtyhyciydkkigdeqekrhltrtllsfgrlqdfteinrpqewkaltkdldyketsnqpfiskttphyhitdnkigfrlgtskelypsleikdganriakypynsgfvahafisvhellplmfyqhltgksedllketvrhiqriykdfeeerintiedlekanqgrlplgafpkqmlgllqnkqpdlsekakikiekliaetkllshrintklksspklgkrrekliktgvladwlvkdfmrfqpvaydaqnqpiksskanstefwfirralalyggeknrlegyfkqtnligntnphpflnkfnwkacrnlvdfyqqylegrekfleaikhqpwepyqyclllkvpkenrknlvkgweqggislprglfteairetlskdltlskpirkeikkhgrvgfisraitlyfkekyqdkhqsfynlsykleakapllkkeehyeywqqnkpqsptesqrlelhtsdrwkdyllykrwqhlekklrlyrnqdimlwlmtleltknhfkelnlnyhqlklenlavnvqeadaklnpinqtlpmvlpvkvypttafgevqyhetpirtvyireeqtkalkmgnflcalvkdrringlfsfikeendtqkhpisqlrlrreleiyqslrvdafketlsleekllnkhaslsslenefrtlleewkkkyaassmvtdkhiafiasvrnafchnqypfyketlhapillftvaqptteekdglgiaeallkylreyceivk sqiPrevotella WP_mendkrleesacytlndkhfwaaflnlarhnvyitvnhinktlelknkknqeiiidndqdilaikthpleuritidis 021584635wakvngdlnktdrlrelmikhfpfleaaiysnnkedkeevkeekqakaqsfkslkdclflfleklq(SEQ IDearnyyshykysesskepefeegllekmyntfdasirlykedyqynkdidpekdflchlerkedfnNO: 86)ylftdkdnkgkitkngllffvslflekkdaiwmqqkfrgflcdnrgnkekmthevfcrsrmllpkirlestqtqdwilldmlnelircpkslyerlqgayrekflcvpfdsidedydaeqepfrntivrhqdrfpyfalryfdyneifknlrfqidlgtyhfsiykkliggkkedrhlthklygferiqeftkqnrpdkwqaiikdldtyetsneryisettphyhlenqkigirfrndnndiwpslktngeknekskynldkpyqaeaflsvhellpmmfyylllkmentdndkednevgtkkkgnknnkqekhkieeiienkikdiyalydaftngeinsidelaeqregkdieighlpkqlivilknkskdmaekanrkqkemikdtkkrlatldkqvkgeiedggrnirllksgeiarwlyndmmrfqpvqkdnegkpinnskansteyqmlqrslalynkeekptryfrqvnlikssnphpfledtkweecynilsfyrnylkakikflnklkpedwkknqyflmlkepktnrktivqgwkngfnlprgiftepikewfkrhqndseeykkvealdrvglvakviplffkeeyfkedaqkeinncvqpfysfpynvgnihkpeeknflhceerrklwdkkkdkfkgykakekskkmtdkekeehrsylefqswnkferelrlyrnqdiltwllctklidklkidelnieelqklrlkdidtdtakkeknnilnrvmpmrlpvtvyeidksfnivkdkplhtvyieetgtkllkqgnfkalvkdrrlnglfsfvktsseaeskskpisklrveyelgayqkaridiikdmlalektlidndenlptnkfsdmlkswlkgkgeankarlqndvgllvavrnafshnqypmynsevfkgmkllslssdipekeglgiakqlkdkiketieriieiekeirn Porphyromonas WP_mntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllcdhllsvgingivalis 021663197drwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslldflrndfshnrldgttf(SEQ IDehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrkeqlisvadgkecltvsglafficlflNO: 87)dreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnprsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmdqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlqkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrhqfraivaelrlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkykdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpi ndlPorphyromonas WP_mntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllcdhllsvgingivalis 021665475drwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslldflrndfshnrldgttf(SEQ IDehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrkeqlisvadgkecltvsglafficlflNO: 88)dreqasgmlsrirgfkrtnenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsgflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkykdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildkenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildhenrffgkllnnmsqpi ndlPorphyromonas WP_mntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllcdhllsvgingivalis 021677657drwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslldflrndfshnrldgttf(SEQ IDehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrkeqlisvadgkecltvsglafficlflNO: 89)dreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsgflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkykdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildhenrffgkllnnmsqpi ndlPorphyromonas WP_mntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllcdhllsvgingivalis 021680012drwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslldflrndfshnrldgttf(SEQ IDehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrkeqlisvadgkecltvsglafficlflNO: 90)dreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnprsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmdqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlqkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrhqfraivaelrlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskvmellkykdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvrdkkrelrtagkpvppdlaayikrsfhravnerefmlrlvqeddrlmlmainkimtdreedilpglknidsildkenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrypglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaeipliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpindlPorphyromonas WP_mntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllcdhllsvgingivalis 023846767drwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslldflrndfshnrldgttf(SEQ IDehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrkeqlisvadgkecltvsglafficlflNO: 91)dreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnprsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkykdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslypatieikskrkdwskyiryrydrrypglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpindlPrevotella WP_mkndnnstkstdytlgdkhfwaaflnlarhnvyitvnhinkvlelknkkdqeiiidndqdilaiktlfalsenii 036884929wgkvdtdinkkdrlrelimkhfpfleaatyqqsstnntkqkeeeqakaqsfeslkdclflfleklre(SEQ IDarnyyshykhsksleepkleekllenmynifdtnvqlvikdyehnkdinpeedflchlgraegefnNO: 92)yyftrnkkgnitesgllffvslflekkdaiwaqtkikgflcdnrenkqkmthevfcrsrmllpklrlestqtqdwilldmlnelircpkslykrlqgekrekfrvpfdpadedydaeqepflcntivrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkqigdkkedrhlthklygferiqefakenrpdewkalvkdldtfeesnepyisettphyhlenqkigirnknkkkkktiwpsletkttvnerskynlgksfkaeaftsvhellpmmfyylllnkeepnngkinaskvegiiekkirdiyklygafaneeinneeelkeycegkdiairhlpkqmiailkneykdmakkaedkqkkmikdtkkrlaaldkqvkgevedggrnikplksgriaswlyndmmrfqpvqrdrdgypinnskansteyqllqrtialfgsererlapyfrqmnligkdnphpflkdtkwkehnnilsfyrsyleakknflgslkpedwkknqyflklkepktnretivqgwkngfnlprgiftepirewfirhqneseeykkvkdfdriglvakviplifkedyqkeiedyvqpfygypfnvgnihnsqegifinkkereelwkgnktkflcdyktkeknkektnkdkfkkktdeekeefrsyldfqswkkferelrlvrnqdivtwilcmelidklkidelnieelqklrlkdidtdtakkeknnilnrimpmelpvtvyetddsnniikdkplhtiyikeaetkllkqgnflcalvkdrringlfsfvetsseaelkskpiskslveyelgeyqrarveiikdmirleetligndeklptnkfrqmldkwlehkketddtdlkndvklltevrnafshnqypmrdriafanikpfslssantsneeglgiakklkdktketidriieieeqtatkr Prevotella WP_mendkrleestcytlndkhfwaaftnlarhnvyitinhinklleirqidndekvldikalwqkvdkpleuritidis 036931485dinqkarlrelmikhfpfleaaiysnnkedkeevkeekqakaqsfkslkdclflfleklqearnyys(SEQ IDhykssesskepefeegllekmyntfgvsirlykedyqynkdidpekdfichlerkedfnylftdkdNO: 93)nkgkitkngliffvslflekkdaiwmqqklrgfkdnrgnkekmthevfcrsrmllpkirlestqtqdwilldmlnelircpkslyerlqgayrekfkvpfdsidedydaeqepfrntivrhqdrfpyfalryfdyneifknlrfqidlgtyhfsiykkligdnkedrhlthklygferiqefakqkrpnewqalvkdldiyetsneqyisettphyhlenqkigirfknkkdkiwpsletngkenekskynldksfqaeaftsihellpmmfydillkkeepnndeknasivegfikkeikrmyaiydafaneeinskegleeycknkgfqerhlpkqmiailtnksknmaekakrkqkemikdtkkrlatldkqvkgeiedggrnirllksgeiarwlyndmmrfqsvqkdkegkpinnskansteyqmlqrslalynkeqkptpyfiqvnlikssnphpfleetkweecnnilsfyrsyleakknfleslkpedwkknqyflmlkepktnrktivqgwkngfnlprgiftepikewfkrhqndseeykkvealdrvglvakviplfficeeyficedaqkeinncvqpfysfpynvgnihkpeeknflhceerrklwdkkkdkfkgykakekskkmtdkekeehrsylefqswnkferelrivrnqdivtwilctelidklkidelnieelqklrlkdidtdtakkeknnilnrimpmqlpvtvyeidksfnivkdkplhtiyieetgtkllkqgnfkalvkdrringlfsfvktsseaeskskpisklryeyelgayqkaridiikdmlalektiidndenlptnkfsdmlkswlkgkgeankarlqndvdllvairnafshnqypmynsevfkgmkllslssdipekeglgiakqlkdkiketieriieiekeirn[Porphyromonas WP_mtegnerpyngtyytiedkhfwaaffnlarhnayitiahidrqlayskaditndedilfflcgqwkngingivalis 039417390ldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekeelqanalsldnlksilf(SEQ IDdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlNO: 94)vrkgkkdrygnndnpffichhfvdregtvteagliffvslflekrdaiwmqkkirgflcggteayqqmtnevfcrsrislpklkleslrtddwmildmlnelvrcpkslydrlreedrarfrvpidilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrivrdldyfetgdkpyitqttphyhiekgkiglrfvpegqhlwpspevgatrtgrskyaqdkrltaeaflsvhelmpmmfyyfilrekyseevsaekvqgrikrviedvyavydafargeidtldrldacladkgirrghlprqmiailsqehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkafiqsigrsdreenhrifilkepktdrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyldfkswqkferelrlyknqdiitwmmcrdlmeenkvegldtgtlylkdirtdvheqgslnylnrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnflcsfvkdrringlfsfvdtgalameqypisklrveyelakyqtarvcafeqtleleeslltryphlpdknfrkmleswsdplldkwpdlhrkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqakemaeriiqv PorphyromonasWP_ mteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsflcalwknlgulae 039418912dndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekeelqanalsldnlksilfd(SEQ IDflqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlvNO: 95)rkgkkdryghndnpsflchhfvdsegmvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrmddwmlldmlnelvrcpkplydrlreddracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldyfetgdkpyisqtsphyhiekgkiglrfmpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldacladkgirrghlpkqmiailsgehknmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdasgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhdtrweshtnilsfyrsylrarkaflerigrsdrmenrpflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskeeraeewergkerfrdleawshsaarriedafagieyaspgnkkkieqllrdlslweafesklkvradkinlaklkkeileaqehpyhdflcswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirtnvqeqgslnylnhvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglameqypisklrveyelakyqtarvcafeqtleleeslltryphlpdknfrkmleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa Porphyromonas WP_mteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsflcalwknlgulae 039419792dndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekeelqanalsldnlksilfd(SEQ IDflqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlvNO: 96)rkgkkdryghndnpsflchhfvdgegmvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlrekdrarfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkvigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldyfetgdkpyisqttphyhiekgkiglrfvpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintrdeldacladkgirrghlpkqmigilsqehknmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpfldetrweshtnilsfyrsylrarkaflerigrsdrvenrpflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdryqpfydspfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileaqehpyhdfkswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirpnvqeqgslnvinrvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglameqypisklryeyelakyqtarvcvfeltlrleesllsryphlpdesfremleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa PorphyromonasWP_ mteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsflcalwknfgulae 039426176dndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekeelqanalsldnlksilfd(SEQ ID flqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphyhfnhlNO: 97)vrkgkkdryghndnpsflchhfvdsegmvteagllffvslflekrdaiwmqkkirgfkggtgpyeqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlrekdracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldyfetgdkpyisqttphyhiekgkiglrfmpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrvikdvyaiydafardeintlkeldacsadkgirrghlpkqmigilsqehknmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpfldetrweshtnilsfyrsylrarkaflerigrsdrvenrpflllkepkndrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdrvqpfydspfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyhdfkswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirtdvheqgslnvinrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnflcsfvkdrrlnglfsfvdtgglameqypisklrveyelakyqtarvcafeqtleleeslltryphlpdenfremleswsdpllgkwpdlhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa PorphyromWP_ mteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsflcalwknfonas gulae 039431778dndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekeelqanalsldnlksilfd(SEQ IDflqklkdfrnyyshyrhsesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlvNO: 98)rkgkkdryghndnpsflchhfvdgegmvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlreddracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldyfetgdkpyisqtsphyhiekgkiglrfmpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldacladkgirrghlpkqmiailsgehkdmeekirkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkplnnskansteyrmlqralalfggekkrltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrmenrpflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskeeraeewergkerfrdleawshsaarriedafagieyaspgnkkkieqllrdlslweafesklkvradkinlaklkkeileaqehpyhdflcswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirpnvqeqgslnylnrvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrrlnglfsfvdtgglameqypisklrveyelakyqtarvcvfeltlrleeslltryphlpdesfrkmleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqv Porphyromonas WP_mteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndedilffkgqwknlgulae 039437199dndlerksrlrslilkhfsflegaaygkkffeskssgnkssknkeltkkekeelqanalsldnlksilf(SEQ ID dflqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkrdhehndevdphyhfnhNO: 99)lvrkgkkdryghndnpsflchhfvdgegmvteagllffvslflekrdaiwmqkkirgflcggtepyeqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlrekdracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldyfetgdkpyisqttphyhiekgkiglrfvpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldacladkgirrghlpkqmigilsqerkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkplnnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrvencpflllkepktdrqtivagwkgefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileaqehpyhdfkswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirpnvqeqgslnvinrvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrrlnglfsfvdtgalameqypiskirveyelakyqtarvcafeqtleleeslltryphlpdesfremleswsdplltkwpelhgkvffliavrnafshnqypmydeavfssiwkydpsspdaieermglniahrlseevkqaketieriiqaPorphyromonas WP_mteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsflcalwknlgulae 039442171dndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekeelqanalsldnlksilfd(SEQ ID flqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphyhfnhlNO: 100)vrkgkkdryghndnpsflchhfvdsegmvteagllffvslflekrdaiwmqkkirgfkggtgpyeqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlrekdracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldyletgdkpyisqttphyhiekgkiglrfvpegqhlwpspevgttrtgrskcaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldtcladkgirrghlpkqmitilsgerkdmkekirkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdasgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrvencpflllkepktdrqtivagwkdefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedryqpfydspfnvgnslkpkkgrflskedraeewergmerfrdleawshsaarrikdafagieyaspgnkkkieqllrdlslweafesklkvradkinlaklkkeileaqehpyhdflcswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirpnvqeqgslnylnrvkpmrlpvvvyradsrghvhkeaplatvyieerntkllkqgnfksfvkdrringlfsfvdtgglameqypisklrveyelakyqtarycvfeltlrleesllsryphlpdesfremleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa Porphyromonas WP_mntvpatenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllcdhllsidgulae 039445055rwtkvyghsrrylpflhcfdpdsgiekdhdsktgvdpdsaqrlirelyslldflrndfshnrldgttfe(SEQ IDhlkvspdissfitgaytfaceraqsrfadffkpddfllaknrkeqlisvadgkecltvsgfafficlfldrNO: 101)eqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkykdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvrdkkrelrtagkpvppdlaayikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildhenrffgkllnnmsqpindlCapnocyto WP_menktslgnniyynpfkpqdksyfagylnaamenidsvfrelgkrlkgkeytsenffdaifkeni phaga041989581slveyeryvkllsdyfpmarlldkkevpikerkenflcknfrgiikavrdlrnfythkehgeveitdecynodegmi (SEQ IDifgvldemlkstvltykkkkiktdktkeilkksiekqldilcqkkleylkdtarkieekrrnqrergekNO: 102)klvprfeysdrrddliaaiyndafdvyidkkkdslkessktkyntesypqqeegdlkipiskngvvfllslflskqevhafkskiagfkatvideatvshrknsicfmatheifshlaykklkrkvrtaeinyseaenaeqlsiyaketlmmqmldelskvpdvvyqnlsedvqktfiedwneylkenngdvgtmeeeqvihpvirkryedkfnyfairfldefaqfptlrfqvhlgnylhdsrpkehlisdrrikekitvfgrlselehkkalfikntetnedrkhywevfpnpnydfpkenisvndkdfpiagsildrekqptagkigikvnllnqkyisevdkavkahqlkqrnnkpsigniieeivpingsnpkeiivfggqptaylsmndihsilyeffdkwekkkeklekkgekelrkeigkeleekivgkiqtqiqqiidkdinakilkpyqdddstaidkeklikdlkqeqkilqklkneqtarekeyqeciayqeesrkikrsdksrqkylrnqlkrkypevptrkeilyyqekgkvavwlandikrfmptdfknewkgeqhsllqkslayyeqckeelknllpqqkvfkhlpfelgghfqqkylyqfytryldkrlehisglvqqaenfknenkvfkkvenecfkflkkqnythkgldaqaqsvlgypiflergfmdekptiikgktfkgneslftdwfryykeyqnfqtfydtenyplvelekkqadrkretkiyqqkkndvifilmakhiflcsvfkqdsidrfsledlyqsreerlenqekakqtgerntnyiwnktvdlnlcdgkvtvenvklknvgnfikyeydqrvqtflkyeenikwqaflikeskeeenypyivereieqyekvrreellkevhlieeyilekvkdkeilkkgdnqnfkyyilngllkqlknedvesykvfnlntkpedvninqlkqeatdleqkafvltyirnkfahnqlpkkefwdycqekygkiekektyaeyfaevflcrekealmk Prevotella WP_mnipalvenqkkyfgtysvmamlnaqtvldhiqkvadiegeqnennenlwfhpvmshlyna sp. P5-119042518169kngydkqpektmfiierlqsyfpflkimaenqreysngkykqnrvevnsndifevlkrafgvlk (SEQ IDmyrdltnhyktyeeklidgcefltsteqplsgmiskyytvalrntkerygyktedlafiqdnikkitkNO: 103)daygkrksqvntgfflslqdyngdtqkklhlsgvgialliclfldkqyiniflsrlpifssynaqseerriiirsfginsiklpkdrihseksnksvamdmlnevkrcpdelfttlsaekqsrfriisddhnevlmkrstdrfvplllqyidygklfdhirfhvnmgklryllkadktcidgqtrvrvieqpingfgrleeaetmrkqengtfgnsgirirdfenvkrddanpanypyivdtythyilennkvemfisdkgssapllplieddryvvktipscrmstleipamafhmflfgskkteklivdvhnrykrlfqamqkeevtaeniasfgiaesdlpqkildlisgnahgkdvdafirltvddmltdterrikrflcddrksirsadnkmgkrgfkqiStgkladflakdivlfqpsyndgenkitglnyrimqsaiavydsgddyeakqqfklmfekarligkgttephpflykvfarsipanavdfyerylierkfyltglcneikrgnrvdvpfirrdqnkwktpamktlgriysedlpvelprqmfdneikshlkslpqmegidfnnanytyliaeymkrvinddfqtfyqwkrnyhymdmlkgeydrkgslqhcftsveereglwkerasrteryrklasnkirsnrqmrnasseeietildkrlsncrneyqksekvirryrvqdallfllakktlteladfdgerfklkeimpdaekgilseimpmsftfekggkkytitsegmklknygdffvlasdkrignllelvgsdivskedimeefnkydqcrpeissivfnlekwafdtypelsarvdreekvdflcsilkillnnkninkeqsdilrkirnafdhnnypdkgiveikalpeiamsikkafgeyaimk Prevotella WP_mnipalvenqkkyfgtysvmamlnaqtvldhiqkvadiegeqnennenlwfhpvmshlyna sp. P4-76044072147kngydkqpektmfiierlqsyfpflkimaenqreysngkykqnrvevnsndifevlkrafgvlk (SEQ IDmyrdqashyktydeklidgcefltsteqplsgminnyytvalrnmnerygyktedlafiqdkrfkfNO: 104)vkdaygkkksqvntgfflslqdyngdtqkklhlsgvgialliclfldkqyiniflsrlpifssynaqseerriiirsfginsikqpkdrihseksnksvamdmlneikrcpnelfetlsaekqsrfriisndhnevlmkrssdrfvplllqyidygklfdhirfhvnmgklryllkadktcidgqtrvrvieqpingfgrleevetmrkqengtfgnsgirirdfenmkrddanpanypyivdtythyilennkvemfisdeetpapllpvieddryvvktipscrmstleipamafhmflfgskkteklivdvhnrykrlfkamqkeevtaeniasfgiaesdlpqkiidlisgnahgkdvdafirltvddmladterrikrfkddrksirsadnkmgkrgfkqistgkladflakdivlfqpsvndgenkitglnyrimqsaiavynsgddyeakqqfklmfekarligkgttephpflykvfvrsipanavdfyerylierkfyliglsneikkgnrvdvpfirrdqnkwktpamktlgriydedlpvelprqmfdneikshlkslpqmegidfnnanytyliaeymkrvinddfqtfyqwkrnyrymdmlrgeydrkgslqscftsveereglwkerasrteryrklasnkirsnrqmrnasseeietildkrlsnsrneyqksekvirryrvqdallfllakktlteladfdgerfklkeimpdaekgilseimpmsftfekggkkytitsegmklknygdffvlasdkrignllelvgsdtvskedimeefkkydqcrpeissivfnlekwafdtypelsarvdreekvdflcsilkillnnkninkeqsdilrkirnafdhnnypdkgvveiralpeiamsikkafgeyaimk Prevotella WP_mnipalvenqkkyfgtysvmamlnaqtvldhiqkvadiegeqnennenlwfhpvmshlyna sp. P5-60044074780kngydkqpektmfiierlqsyfpflkimaenqreysngkykqnrvevnsndifevlkrafgvlk (SEQ IDmyrdltnhyktyeeklidgcefltsteqpfsgmiskyytvalrntkerygykaedlafiqdnrykftNO: 105)kdaygkrksqvntgsflslqdyngdttkklhlsgvgialliclfldkqyinlflsrlpifssynaqseerriiirsfginsikqpkdrihseksnksvamdmlnevkrcpdelfttlsaekqsrfriisddhnevlmkrssdrfvplllqyidygklfdhirfhvnmgklryllkadktcidgqtrvrvieqpingfgrleevetmrkqengtfgnsgirirdfenmkrddanpanypyivetythyilennkvemfisdeenptpllpvieddryvvktipscrmstleipamafhmflfgsektekliidvhdrykrlfqamqkeevtaeniasfgiaesdlpqkimdlisgnahgkdvdafirltvddmltdterrikrfkddrksirsadnkmgkrgfkqistgkladflakdivlfqpsyndgenkitglnyrimqsaiavydsgddyeakqqflclmfekarligkgttephpflykvfvrsipanavdfyerylierkfyliglsneikkgnrvdvpfirrdqnkwktpamktlgriysedlpvelprqmfdneikshlkslpqmegidfnnanytyliaeymkrvinddfqtfyqwkrnyrymdmlrgeydrkgslqhcftsieereglwkerasrteryrklasnkirsnrqmrnasseeietildkrlsncrneyqksekiirryrvqdallfllakktlteladfdgerfklkeimpdaekgilseimpmsftfekggkiytitsggmklknygdffvlasdkrignllelvgsntvskedimeefickydqcrpeissivfnlekwafdtypelparvdrkekvdfwsildvlsnnkdinneqsyilrkirnafdhnnypdkgiveikalpeiamsikkafgeyaimk Phaeo- WP_mtntpkrrtlhrhpsyfgaflniarhnafmimehlstkydmedkntldeaqlpnaklfgclkkrygdactylibacter 044218239kpdvtegvsrdlrryfpflnyplflhlekqqnaeqaatydinpedieftlkgffrllnqmrnnyshyixiamenensis (SEQ IDsntdygkfdklpvqdiyeaaiffildrgkhtkrfdvfeskhtrhlesnnseyrprslanspdhentvaNO: 106)fvtclflerkyafpflsrldcfrstndaaegdplirkashecytmfccrlpqpklessdilldmvnelgrcpsalynllseedqarfhikreeitgfeedpdeeleqeivlkrhsdrfpyfalryfddteafqtlrfdvylgrwrtkpvykkriymerdryltqsirtftrlsrllpiyenvkhdavrqneedgklvnpdvtsqfhkswiqiesddraflsdriehfsphynfgdqviglkfinpdryaaiqnvfpklpgeekkdkdaklvnetadaiistheirslflyhylskkpisagderrfiqvdtetfikqyidtiklffediksgelqpiadppnyqkneplpyvrgdkektqeeraqyrerqkeikerrkelntllqnryglsiqyipsrlreyllgykkvpyeklalqklraqrkevkkrikdiekmrtprvgeqatwlaedivfltppkmhtperkttkhpqklnndqfrimqsslayfsvnkkaikkffqketgiglsnretshpflyridvgrcrgildfytgylkykmdwlddaikkvdnrkhgkkeakkyekylpssiqhktpleldytrlpvylprglfkkaivkalaahadfqvepeednvifcldqlldgdtqdfynwqryyrsalteketdnqlvlahpyaeqilgtiktlegkqknnklgnkakqkikdelidlkrakrrlldreqylravqaedralwlmiqerqkqkaeheeiafdqldlknitkiltesidarlripdtkvditdklplrrygdlrrvakdrrlvnlasyyhvaglseipydlvkkeleeydrrrvaffehvyqfekevydryaaelmenpkgestyfshweyvavavkhsadthfnelfkekvmqlrnkfhhnefpyfdwllpevekasaalyadrvfdvaegyyqkmrklmrq Flavobacterium WP_mdnnitvektelglgitynhdkvedkhyfggffnlaqnnidlvaqeflckrlliqgkdsinifanyfssp. 316 045968377dqcsitnlergikilaeyfpvvsyidldeknksksirehlillletinnlrnyythyyhkkiiidgslfpl(SEQ IDldtillkvvleikkkklkedktkqllkkglekemtilfnlmkaeqkekkikgwnidenikgavinrNO: 107)afshllyndelsdyrkskyntedetlkdtltesgilfllsifinkkeqeqlkanikgykgkiasipdeeitlknnslrnmathwtyshltykglkhriktdheketllvnmvdylskvpheiyqnlseqnkslfledineymrdneenhdsseasrvihpvirkryenkfayfairfldefaefptlrfmvnvgnyihdnrkkdiggtslitnrtikqqinvfgniteihkkkndyfekeenkektlewelfpnpsyhfqkenipifidleksketndlakeyakekkkifgssrkkqqntakknretiinlvfdkyktsdrktvtfeqptallsfnelnsflyaflvenktgkelekiiiekianqyqilkncsstvdktndnipksikkivntttdsfyfegkkidieklekditieiektnekletikeneesaqnykrnerntqkrklyrkyvfftneigieatwitndilrfldnkenwkgyqhselqkfisqydnykkealgllesewnlesdaffgqnlkrmfqsnstfetfykkyldnrkntletylsaienlktmtdvrpkvlkkkwtelfrffdkkiyllstietkinelitkpinlsrgifeekptfingknpnkennqhlfanwfiyakkqtilqdfynlpleqpkaitnlkkhkyklersinnlkiediyikqmvdflyqklfeqsfigslqdlytskekreiekgkakneqtpdesfiwkkqveinthngriiaktkikdigkflailltdnkiahlisyddriwdfslnndgditkklysintelesyetirrekllkqiqqfeqfllegeteysaerkhpekfekdcnpnflckyiiegvinkiipnheieeieilkskedvfkinfsdililnndnikkgyllimirnkfahnqlidknlfnfslqlysknenenfseylnkvcqniiqefkeklkPorphyromonas WP_mteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsflcalwknfgulae 046201018dndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekeelqanalsldnlksilfd(SEQ IDflqklkdfrnyyshyrhsesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlvNO: 108)rkgkkdryghndnpsflchhfvdsegmvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlrekdrarfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldyfetgdkpyisqttphyhiekgkiglrfmpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldacladkgirrghlpkqmiailsgehkdmeekirkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekkrltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrmenrpflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskeeraeewergkerfrdleawshsaarriedafagieyaspgnkkkieqllrdlslweafesklkvradkinlaklkkeileaqehpyhdflcswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirpnvqeqgslnylnrvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglameqypisklrveyelakyqtarvcvfeltbleeslltryphlpdesfrkmleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqv WP_ Chryseo-metqfighgiaydhskiqdkhffggfinlaennikavlkafsekfnvgnvdvkqfadvslkdnlp047431796 bacteriumdndfqkrvsflkmyfpvvdfinipnnrakfrsdlttlfksvdqlrnfythyyhkpldfdaslfillddisp. fartakevrdqkmkddktrqllskslseelqkgyelqlerlkelnrlgkkvnihdqlgikngvinnaYR477fnhliykdgesfktkltyssaltsfesaengieisqsgllfllsmflkrkeiedlknrnkgfkakvvide(SEQ IDdgkvnglkfmathwvfsylcflcglksklstefheetlliqiidelskvpdelycafdketrdkfiediNO: 109)neyvkeghqdfsledakvihpvirkryenkfnyfairfldefvkfpslrfqvhvgnyvhdrriknidgttfetervvkdrikvfgrlseissykaqylssysdkhdetgweifpnpsyvfinnnipihisvdtsfkkeiadfkklrraqvpdelkirgaekkrkfeitqmigsksvinqeepiallslneipallyeilingkepaeieriikdklnerqdviknynpenwlpasqisrrlrsnkgeriintdkllqlvtkellvteqklkiisdnrealkqkkegkyirkfiftnselgreaiwladdikrfmpadvrkewkgyqhsqlqqslafynsrpkealailesswnlkdekiiwnewilksftqnkffdafyneylkgrkkyfaflsehivqytsnaknlqkfikqqmpkdlfekrhyiiedlqteknkilskpfifprgifdkkptfikgvkvedspesfanwyqygyqkdhqfqkfydwkrdysdvflehlgkpfinngdrrtlgmeelkeriiikqdlkikkikiqdlflrliaenlfqkvflcysaklplsdfyltqeermekenmaalqnvreegdkspniikdnfiwskmipykkgqiienavklkdigklnvlslddkvqtllsyddakpwskialenefsigensyevirreklfkeiqqfeseilfrsgwdginhpaqlednrnpkfkmyivngilrksaglysqgediwfeynadfnnldadvletkselvqlaflvtairnkfahnqlpakefyfyirakygfadepsvalvylnftkyainefkkvmi Riemerella WP_mffsfhnaqrvifkhlykafdaslrmykedykahftvnitrdfahlnrkgknkqdnpdfnryrfeanatipestifer 049354263kdgfftesgllfftnlfldkrdaywmlkkvsgfkashkqrekmttevfcrsrillpklrlesrydhnq(SEQ IDmlldmlselsrcpkllyeklseenkkhfqveadgfldeieeeqnpfkdtlirhqdrfpyfalryldlnNO: 110)esflcsirfqvdlgtyhyciydkkigdeqekrhltrtllsfgrlqdfteinrpqewkaltkdldyketsnqpfiskttphyhitdnkigfrlgtskelypsleikdganriakypynsgfvahafisvhellplmfyqhltgksedllketvrhiqriykdfeeerintiedlekanqgrlplgafpkqmlgllqnkqpdlsekakikiekliaetkllshrintklksspklgkrrekliktgvladwlvkdfmrfqpvaydaqnqpiksskanstefwfirralalyggeknrlegyfkqtnligntnphpflnkfnwkacrnlvdfyqqylegrekfleaiknqpwepyqyclllkipkenrknlvkgweqggislprglfteairetlsedlmlskpirkeikkhgrvgfisraitlyfkekyqdkhqsfynlsykleakapllkreehyeywqqnkpqsptesqrlelhtsdrwkdyllykrwqhlekklrlyrnqdvmlwlmtleltknhfkelnlnyhqlklenlavnvqeadaklnpinqtlpmvlpvkvypatafgevqyhktpirtvyireehtkalkmgnflcalvkdrringlfsfikeendtqkhpisqlrlrreleiyqslrydaflcetlsleekllnkhtslsslenefralleewkkeyaassmvtdehiafiasvrnafchnqypfykealhapiplftvaqptteekdglgiaeallkvlreyceivksqi Porphyromonas WP_mteqnekpyngtyytledkhfwaaffnlarhnayitlahidrqlayskaditndedilfflcgqwkngingivalis 052912312ldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekeelqanalsldnlksilf(SEQ IDdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlNO: 111)vrkgkkdkygnndnpfflchhfvdreekvteagllffvslflekrdaiwmqkkirgfkggteayqqmtnevfcrsrislpklkleslrtddwnilldmlnelvrcpkllydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyitqttphyhiekgkiglrfvpegqllwpspevgatrtgrskyaqdkrftaeaflsvhelmpmmfyyfllrekyseeasaekvqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmiailsqehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdreenhrflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdryqpfydypfnvgnslkpkkgrflskekraeewesgkerfrdleawshsaarriedafvgieyaswenkkkieqllqdlslwetfesklkvkadkiniaklkkeileakehpyhdfkswqkferelrlvknqdiitwmmerdlmeenkvegldtgtlylkdirtdvqeqgslnylnhvkpmflpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalameqypisklrveyelakyqtarvcafeqtleleeslltryphlpdesfremleswsdplldkwpdlqrevrlliavrnafshnqypmydetifssirkydpssldaieermglniahrlseevklakemveriiqa Porphyromonas WP_mteqnekpyngtyytlkdkhfwaaffnlarhnayitlthidrqlayskaditndedilffkgqwkngingivalis 058019250ldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekeelqanalsldnlksilf(SEQ ID dflqklkdfrnyyshyrhpesselpmfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnNO: 112)hlvrkgkkdregnndnpfflchhfvdregkvteagllffvslflekrdaiwmqkkirgflcggtetyqqmtnevfcrsrislpklkleslrtddwnilldmlnelvrcpkslydrlreedracfrvpvdilsdeddtdgaeedpflcntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlyrdldcfetgdkpyitqttphyhiekgkiglrfvpegqhlwpspevgatrtgrskyaqdkrftaeaflsvhelmpmmfyyfllrekyseevsaervqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmiailsqkhkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdrvenhrflllkepktdrqtivagwkgefhlprgifteavrdcliemgldevgsykevgfmakavplyferackdryqpfydypfnvgnslkpkkgrflskekraeewesgkerfrdleawshsaarriedafagienasrenkkkieqllqdlslwetfesklkvkadkiniaklkkeileakehpyldflcswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirtdvqeqgslnylnhvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalameqypisklrveyelakyqtarycafeqtleleeslltryphlpdenfrkmleswsdplldkwpdlhrkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqakemaeriiqa Flavobacterium WP_mssknesynkqktfnhykqedkyffggfinnaddnlrqvgkefktrinfnhnnnelasvfkdyfcolumnare 060381855nkeksvakrehalnllsnyfpvleriqkhtnhnfeqtreifellldtikklrdyythhyhkpitinpkv(SEQ IDydflddtlldvlitikkkkvkndtsrellkekfrpeltqlknqkreelikkgkklleenlenavfnhclrNO: 113)pfleenktddkqnktvslrkyrkskpneetsitltqsglvflisifihrkefqvftsglegfkakvntikeeeislnknnivymithwsysyynfkglkhriktdqgvstleqnntthsltntntkealltqivdylskvpneiyetlsekqqkefeedineymrenpenedstfssivshkvirkryenkfnyfamrfldeyaelptlrfmvnfgdyikdrqkkilesiqfdseriikkeihlfeklglvteykknvylketsnidlsrfplfpspsyvmannnipfyidsrsnnldeylnqkkkaqsqnrkrnitfekynkeqskdaiiamlqkeigykdlqqrstigllscnelpsmlyevivkdikgaelenkiaqkireqyqsirdffldspqkdnipttltktistdtsvtfenqpidiprlknalqkeltitqeklinvkqheievdnynrnkntykfknqpkdkvddnklqrkyvfyrneigqeanwlasdlihfmknkslwkgymhnelqsflaffedkkndcialletvfnlkedciltkdlknlflkhgnfidfykeylklkedflntestflengfiglppkilkkelskrinyifivfqkrqfiikeleekknnlyadainlsrgifdekptmipflckpnpdefaswfvasyqynnyqsfyeltpdkiendkkkkyknlrainkvkiqdyylklmvdtlyqdlfnqpldkslsdfyvsktdrekikadakayqkrndsflwnkvihlslqnnritanpklkdigkykralqdekiatlltyddrtwtyalqkpekenendykelhytalnmelqeyekvrskkllkqvqelekqildkfydfsnnathpedleiedkkgkrhpnfklyitkallkneseiinlenidieilikyydynteklkekiknmdedekakivntkenynkitnvlikkalvliiirnkmahnqyppkfiydlatrfvpkkeeeyfacyfnrvfetittelwenkkkake ivPorphyromonas WP_mteqnerpyngtyytledkhfwaaffnlarhnayitlthidrqlayskaditndedilffkgqwknlgingivalis 061156470dndlerkarlrslilkhfsflegaaygkklfenkssgnksskkkeltkkekeelqanalsldnlksilf(SEQ IDdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlNO: 114)vrkgkkdregnndnpffichhfvdregkvteagliffvslflekrdaiwmqkkirgflcggteayqqmtnevfcrsrislpklkleslrtddwmildmlnelvrcpkslydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrivrdldyfetgdkpyitqttphyhiekgkiglrfvpegqhlwpspevgatrtgrskyaqdkrltaeaflsvhelmpmmfyyfilrekyseevsaekvqgrikrviedvyavydafargeidtldrldacladkgirrghlprqmiailsqehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkafiqsigrsdreenhrifilkepktdrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyldfkswqkferelrlyknqdiitwmmerdlmeenkvegldtgtlylkdirtevqeqgslnvinrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglameqypisklrveyelakyqtarvcafeqtleleeslltrcphlpdknfrkmleswsdplldkwpdlqrevwlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqakemaeriiqa PorphyromonasWP_ mntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqslicdhilsvgingivalis 061156637drwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslldfirndfshnridgttf(SEQ IDehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrkeqlisvadgkecltvsglafficlflNO: 115)dreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallidmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrliwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsgflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildkenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpi ndlRiemerella WP_mffsfhnaqrvifkhlykafdaslrmvkedykahftvnitrdfahlnrkgknkqdnpdfnryrfeanatipestifer 061710138kdgfftesgllfftnlfldkrdaywmlkkvsgfkashkqsekmttevfcrsrillpklrlesrydhnq(SEQ IDmlldmlselsrcpkllyeklsekdkkcfqveadgfldeieeeqnpfkdtlirhqdrfpyfalryldlnNO: 116)esfksirfqvdlgtyhyciydkkigyeqekrhltrtllnfgrlqdfteinrpqewkaltkdldynetsnqpfiskttphyhitdnkigifirtskelypslevkdganriakypynsdfvahafisisvhellplmfyqhltgksedllketvrhiqriykdfeeerintiedlekanqgrlplgafpkqmlgllqnkqpdlsekakikiekliaetkllshrintklksspklgkrrekliktgvladwlvkdfmrfqpvvydaqnqpiksskanstesrlirralalyggeknrlegyfkqtnligntnphpflnkfnwkacrnlvdfyqqylegekfleaikhqpwepyqyclllkvpkenrknlvkgweqggislprglfteairetlskdltlskpirkeikkhgrvgfisraitlyfkekyqdkhqsfynlsykleakapllkkeehyeywqqnkpqsptesqrlelhtsdrwkdyllykrwqhlekklrlyrnqdimlwlmtleltknhfkelnlnyhqlklenlavnvqeadaklnpinqtlpmvlpvkvypttafgevqyhetpirtvyireeqtkalkmgnfkalvkdrhlnglfsfikeendtqkhpisqlrlrreleiyqslrydafketlsleekllnkhaslsslenefrtlleewkkkyaassmvtdkhiafiasvrnafchnqypfyketlhapillftvaqptteekdglgiaeallrylreyceivksqi Flavobacterium WP_mssknesynkqktfnhykqedkyffggflnnaddnlrqvgkefktrinfnhnnnelasvfkdyfcolumnare 063744070nkeksvakrehalnllsnyfpvleriqkhtnhnfeqtreifellldtikklrdyythhyhkpitinpki(SEQ IDydflddtlldvlitikkkkvkndtsrellkeklrpeltqlknqkreelikkgkklleenlenavfnhclrNO: 117)pfleenktddkqnktvslrkyrkskpneetsitltqsglvflmsifihrkefqvftsglegfkakvntikeekislnknnivymithwsysyynfkglkhriktdqgvstleqnntthsltntntkealltqivdylskvpneiyetlsekqqkefeedineymrenpenedstfssivshkvirkryenkfnyfamrfldeyaelptlrfmvnfgdyikdrqkkilesiqfdseriikkeihlfeklglvteykknvylketsnidlsrfplfpspsyvmannnipfyidsrsnnldeylnqkkkaqsqnrkrnitfekynkeqskdaiiamlqkeigvkdlqqrstigllscnelpsmlyevivkdikgaelenkiaqkireqyqsirdftlnspqkdnipttliktistdtsvtfenqpidiprlknaiqkelaltqekllnvkqheievnnynrnkntykfknqpkdkvddnklqrkyvfyrneigqeanwlasdlihfmknkslwkgymhnelqsflaffedkkndcialletvfnlkedciltkdlknlflkhgnfidfykeylklkedflntestflengfiglppkilkkelskrinyifivfqkrqfiikeleekknnlyadainlsrgifdekptmipfkkpnpdefaswfvasyqynnyqsfyeltpdkiendkkkkyknlrainkvkiqdyylklmvdtlyqdlfnqpldkslsdfyvsktdrekikadakayqkrndsflwnkvihlslqnnritanpklkdigkykralqdekiatlltyddrtwtyalqkpekenendykelhytalnmelqeyekvrskkllkqvqelekqildkfydfsnnathpedleiedkkgkrhpnfklyitkallkneseiinlenidieilikyydynteklkekiknmdedekakivntkenynkitnvlikkalvliiirnkmahnqyppkfiydlatrfvpkkeeeyfacyfnrvfetittelwenkkkakeiv Riemerella WP_mekplppnvytlkhkffwgaflniarhnafitichineqlglttppnddkiadvvcgtwnnilnndanatipestifer 064970887hdllkksqltelilkhfpflaamcyhppkkegkkkgsqkeqqkekeneaqsqaealnpselikvl(SEQ IDktivkqlrtlrnyyshhshkkpdaekdifkhlykafdaslrmvkedykahftvnitqdfahlnrkgNO: 118)knkqdnpdfdryrfekdgfftesgllfftnlfldkrdaywmlkkvsgfkashkqsekmttevfcrsrillpklrlesrydhnqmlldmlselsrypkllyeklseedkkrfqveadgfldeieeeqnpfkdtlirhqdrfpyfalryldlnesfksirfqvdlgtyhyciydkkigdeqekrhltrtllsfgrlqdfteinrpqewkaltkdldyketskqpfiskttphyhitdnkigifigtskelypslevkdganriaqypynsdfvahafisvhellplmfyqhltgksedllketvrhiqriykdfeeerintiedlekanqgrlplgafpkqmlgllqnkqpdlsekakikiekliaetkllshrintklksspklgkrrekliktgvladwlvkdfmrfqpvaydaqnqpiesskanstefqliqralalyggeknrlegyfkqtnligntnphpflnkfnwkacrnlvdfyqqylecrekfleaiknqpwepyqyclllkipkenrknlvkgweqggislprglfteairetlskdltlskpirkeikkhgrvgfisraitlyfrekyqddhqsfydlpykleakasplpkkehyeywqqnkpqsptelqrlelhtsdrwkdyllykrwqhlekklrlyrnqdvmlwlmtleltknhfkelnlnyhqlklenlavnvqeadaklnpinqtlpmvlpvkvypatafgevqyqetpirtvyireeqtkalkmgnfkalvkdrringlfsfikeendtqkhpisqlrlrreleiyqslrvdafketlnleekllkkhtslssvenkfrilleewkkeyaassmvtdehiafiasvrnafchnqypfyeealhapiplftvaqqtteekdglgiaeallrvlreyceivksqi Sinomicrobium WP_mestttlglhlkyqhdlfedkhyfgggvnlavqniesifqafaerygiqnplrkngvpainnifhdoceani 072319476.1nisisnykeylkflkqylpvvgfleksneinifefredfeilinaiyklrhfythyyhspikledrfytc(SEQ IDlnelfvavaiqvkkhkmksdktrqllnknlhqllqqlieqkreklkdkkaegekvsldtksienavNO: 119)lndafvhlldkdenirinyssrlsediitkngitlsisgllfllslflqrkeaedlrsriegfkgkgnelrfmathwvfsylnykrikhrintdfqketlliqiadelskvpdevyktldhenrskfledineyiregnedaslnestvvhgvirkryenkfhylvlryldefvdfpslrfqvhlgnyihdrrdkvidgtnfitnrvikepikvfgklshvsklksdymeslsrehkngwdvfpnpsynfvghnipifinlrsasskgkelyrdlmkiksekkkksreegipmerrdgkptkieisnqidrnikdnnflcdiypgeplamlslnelpallfellrrpsitpqdiedrmveklyerfqiirdykpgdglstskiskklrkadnstrldgkkllraiqtetrnareklhtleenkalqknrkrrtvyttreqgreaswlaqdlkrfmpiasrkewrgyhhsqlqqilafydqnpkqplelleqfwdlkedtyvwnswihkslsqhngfvpmyegylkgrlgyykklesdiigfleehkvlkryytqqhlnvifrerlyfiktetkqklellarplvfprgifddkptfvqdkkvvdhpelfadwyvysykddhsfqefyhykrdyneifetelswdidfkdnkrqlnpseqmdlfrmkwdlkikkikiqdiflkivaediylkifghkiplslsdfyisrqerltldeqavaqsmrlpgdtsenqikesnlwqttvpyekeqirepkiklkdigkfkyflqqqkvinllkydpqhvwtkaeleeelyigkhsyevvrremllqkchqlekhileqfrfdgsnhpreleqgnhpnfkmyivngiltkrgeleieaenwwlelgnsknsldkvevelltmktipeqkafllilirnkfahnqlpadnyfhyasnlmnlkksdtyslfwftvadtivqefmsl Reichen- WP_mktnpliassgekpnykkfntesdksfkkifqnkgsiapiaekacknfeikskspvnrdgrlhyfsbachiella 073124441.1vghafknidsknyfryeldesqmdmkptqflalqkeffdfqgalngllkhirnvnshyvhtfeklagariperforans (SEQ IDeiqsinqklitflieafelavihsylneeelsyeaykddpqsgqklvqflcdkfypnkeheveerktiNO: 120)laknkrqalehllfievtsdidwklfekhkvftisngkylsfhaclfllslflykseanqliskikgfkrnddnqyrskrqiftffskkftsqdvnseeqhlvkfrdviqylnhypsawnkhlelksgypqmtdklmryiveaeiyrsfpdqtdnhrfllfaireffgqscldtwtgntpinfsnqeqkgfsyeintsaeikdietklkalvlkgpinfkekkeqnflekdkrekkeqptnrvkeklltriqhnmlyvsygrnqdrfmdfaarflaetdyfgkdakfkmyqfytsdeqrdhlkeqkkelpkkefeklkyhqsklvdyftyaeqqarypdwdtpfvvennaiqikvtlfngakkivsvqrnlmlylledalysekrenagkglisgyfvhhqkelkdqldileketeisreqkrefkkllpkrilhryspaqindttewnpmevileeakaqeqryqlllekailhqteedfikrnkgkqfklrfvrkawhlmylkelymnkvaehghhksfhitkeefndfcrwmfafdevpkykeylcdyfsqkgffnnaeflcdliesstslndlyektkqrfegwskdltkqsdenkyllanyesmlkddmlyvnishfisyleskgkinrnahghiaykalnnvphlieeyyykdrlapeeykshgklynklktvkledallyemamhylslepalvpkvktkvkdilssniafdikdaaghhlyhllipfhkidsfvalinhqsqqekdpdktsflakiqpylekvknskdlkavyhyykdtphtlryedlnmihshivsqsvqftkvalkleeyfiakksitlqiarqisyseiadlsnyftdevrntafhfdvpetaysmilqgiesefldreikpqkpkslselstqqvsvctafletlhnnlfdrkddkkerlskareryfeqin

The following Cas13c orthologues were codon optimized for expression inmammalian cells.

TABLE 7 Fusobacterium (SEQMEKFRRQNRNSIIKIIISNYDTKGIKELKVRYRKQAQLDTFIIKTEIVNN necrophorum ID NO:DIFIKSIIEKAREKYRYSFLFDGEEKYHFKNKSSVEIVKKDIFSQTPDNM subsp. 121)IRNYKITLKISEKNPRVVEAEIEDLMNSTILKDGRRSARREKSMTERKL funduliformeIEEKVAKNYSLLANCPMEEVDSIKIYKIKRFLTYRSNMLLYFASINSFL ATCC 51357CEGIKGKDNETEEIWHLKDNDVRKEKVRENFKNKLIQSTENYNSSLK contig00003NQIEEKEKLLRKEFKKGAFYRTIIKKLQQERIKELSEKSLTEDCEKIIKLYSKLRHSLMHYDYQYFENLFENKKNDDLMKDLNLDLFKSLPLIRKMKLNNKVNYLEDGDTLFVLQKTKKAKTLYQIYDALCEQKNGFNKFINDFFVSDGEENTVFKQIINEKFQSEMEFLEKRISESEKKNEKLKKKLDSMKAHFRNINSEDTKEAYFWDIHSSRNYKTKYNERKNLVNEYTELLGSSKEKKLLREEITKINRQLLKLKQEMEEITKKNSLFRLEYKMKIAFGFLFCEFDGNISKFKDEFDASNQEKIIQYHKNGEKYLTSFLKEEEKEKFNLEKMQKIIQKTEEEDWLLPETKNNLFKFYLLTYLLLPYELKGDFLGFVKKHYYDIKNVDFIDENQNNIQVSQTVEKQEDYFYHKIRLFEKNTKKYEIVKYSIVPNEKLKQYFEDLGIDIKYLTVEQKSEVSEEKNKKVSLKNNGMFNKTILLFVFKYYQIAFKLFNDIELYSLFFLREKSGKPLEIFRKELESKMKDGYLNFGQLLYVVYEVLVKNKDLDKILSKKIDYRKDKSFSPEIAYLRNFLSHLNYSKFLDNFMKINTNKSDENKEVLIPSIKIQKMIQFIEKCNLQNQIDFDFNFVNDFYMRKEKMFFIQLKQIFPDINSTEKQKMNEKEEILRNRYHLTDKKNEQIKDEHEAQSQLYEKILSLQKIYSSDKNNFYGRLKEEKLLFLEKQGKKKLSMEEIKDKIAGDISDLLGILKKEITRDIKDKLTEKFRYCEEKLLNLSFYNHQDKKKEESIRVFLIRDKNSDNFKFESILDDGSNKIFISKNGKEITIQCCDKVLETLIIEKNTLKISSNGKIISLIPHYSYSIDVKY Fusobacterium (SEQMEKFRRQNRSSIIKIIISNYDTKGIKELKVRYRKQAQLDTFIIKTEIVNN necrophorum ID NO:DIFIKSIIEKAREKYRYSFLFDGEEKYHFKNKSSVEIVKKDIFSQTPDNM DJ-2 122)IRNYKITLKISEKNPRVVEAEIEDLMNSTILKDGRRSARREKSMTERKL contig0065,IEEKVAENYSLLANCPMEEVDSIKIYKIKRFLTYRSNMLLYFASINSFL whole genomeCEGIKGKDNETEEIWHLKDNDVRKEKVKENFKNKLIQSTENYNSSLK shotgunNQIEEKEKLLRKESKKGAFYRTIIKKLQQERIKELSEKSLTEDCEKIIKL sequenceYSELRHPLMHYDYQYFENLFENKENSELTKNLNLDIFKSLPLVRKMKLNNKVNYLEDNDTLFVLQKTKKAKTLYQIYDALCEQKNGFNKFINDFFVSDGEENTVFKQIINEKFQSEIEFLEKRISESEKKNEKLKKKLDSMKAHFRNINSEDTKEAYFWDIHSSRNYKTKYNERKNLVNEYTELLGSSKEKKLLREEITKINRQLLKLKQEMEEITKKNSLFRLEYKMKMAFGFLFCEFDGNISRFKDEFDASNQEKIIQYHKNGEKYLTYFLKEEEKEKFNLKKLQETIQKTGEENWLLPQNKNNLFKFYLLTYLLLPYELKGDFLGFVKKHYYDIKNVDFMDENQSSKITESKEDDFYHKIRLFEKNTKKYEIVKYSIVPDKKLKQYFKDLGIDTKYLILDQKSEVSGEKNKKVSLKNNGMFNKTILLFVFKYYQIAFKLFNDIELYSLFFLREKSGKPFEVFLKELKDKMIGKQLNFGQLLYVVYEVLVKNKDLSEILSERIDYRKDMCFSAEIADLRNFLSHLNYSKFLDNFMKINTNKSDENKEVLIPSIKIQKMIKFIEECNLQSQIDFDFNFVNDFYMRKEKMFFIQLKQIFPDINSTEKQKMNEKEEILRNRYHLTDKKNEQIKDEHEAQSQLYEKILSLQKIYSSDKNNFYGRLKEEKLLFLEKQEKKKLSMEEIKDKIAGDISDLLGILKKEITRDIKDKLTEKFRYCEEKLLNLSFYNHQDKKKEESIRVFLIRDKNSDNFKFESILDDGSNKIFISKNGKEITIQCCDKVLETLIIEKNTLKISSNGKIISLIPHYSYSIDVKY Fusobacterium (SEQMKVRYRKQAQLDTFIIKTEIVNNDIFIKSIIEKAREKYRYSFLFDGEEKY necrophorum ID NO:HFKNKSSVEIVKNDIFSQTPDNMIRNYKITLKISEKNPRVVEAEIEDLM BFTR-1 123)NSTILKDGRRSARREKSMTERKLIEEKVAENYSLLANCPIEEVDSIKIY contig0068KIKRFLTYRSNMLLYFASINSFLCEGIKGKDNETEEIWHLKDNDVRKEKVKENFKNKLIQSTENYNSSLKNQIEEKEKLSSKEFKKGAFYRTIIKKLQQERIKELSEKSLTEDCEKIIKLYSELRHPLMHYDYQYFENLFENKENSELTKNLNLDIFKSLPLVRKMKLNNKVNYLEDNDTLFVLQKTKKAKTLYQIYDALCEQKNGFNKFINDFFVSDGEENTVFKQIINEKFQSEMEFLEKRISESEKKNEKLKKKLDSMKAHFRNINSEDTKEAYFWDIHSSRNYKTKYNERKNLVNEYTKLLGSSKEKKLLREEITKINRQLLKLKQEMEEITKKNSLFRLEYKMKIAFGFLFCEFDGNISKFKDEFDASNQEKIIQYHKNGEKYLTSFLKEEEKEKFNLEKMQKIIQKTEEEDWLLPETKNNLFKFYLLTYLLLPYELKGDFLGFVKKHYYDIKNVDFMDENQNNIQVSQTVEKQEDYFYHKIRLFEKNTKKYEIVKYSIVPNEKLKQYFEDLGIDIKYLTGSVESGEKWLGENLGIDIKYLTVEQKSEVSEEKNKKVSLKNNGMFNKTILLFVFKYYQIAFKLFNDIELYSLFFLREKSEKPFEVFLEELKDKMIGKQLNFGQLLYVVYEVLVKNKDLDKILSKKIDYRKDKSFSPEIAYLRNFLSHLNYSKFLDNFMKINTNKSDENKEVLIPSIKIQKMIQFIEKCNLQNQIDFDFNFVNDFYMRKEKMFFIQLKQIFPDINSTEKQKKSEKEEILRKRYHLINKKNEQIKDEHEAQSQLYEKILSLQKIFSCDKNNFYRRLKEEKLLFLEKQGKKKISMKEIKDKIASDISDLLGILKKEITRDIKDKLTEKFRYCEEKLLNISFYNHQDKKKEEGIRVFLIRDKNSDNFKFESILDDGSNKIFISKNGKEITIQCCDKVLETLMIEKNTLKISSNGKIISLIPHYSYSIDVKY Fusobacterium (SEQMTEKKSIIFKNKSSVEIVKKDIFSQTPDNMIRNYKITLKISEKNPRVVEA necrophorum ID NO:EIEDLMNSTILKDGRRSARREKSMTERKLIEEKVAENYSLLANCPMEE subsp. 124)VDSIKIYKIKRFLTYRSNMLLYFASINSFLCEGIKGKDNETEEIWHLKD funduliformeNDVRKEKVKENFKNKLIQSTENYNSSLKNQIEEKEKLLRKESKKGAF 1_1_36SYRTIIKKLQQERIKELSEKSLTEDCEKIIKLYSELRHPLMHYDYQYFEN cont1.14LFENKENSELTKNLNLDIFKSLPLVRKMKLNNKVNYLEDNDTLFVLQKTKKAKTLYQIYDALCEQKNGFNKFINDFFVSDGEENTVFKQIINEKFQSEMEFLEKRISESEKKNEKLKKKFDSMKAHFHNINSEDTKEAYFWDIHSSSNYKTKYNERKNLVNEYTELLGSSKEKKLLREEITQINRKLLKLKQEMEEITKKNSLFRLEYKMKIAFGFLFCEFDGNISKFKDEFDASNQEKIIQYHKNGEKYLTYFLKEEEKEKFNLEKMQKIIQKTEEEDWLLPETKNNLFKFYLLTYLLLPYELKGDFLGFVKKHYYDIKNVDFMDENQNNIQVSQTVEKQEDYFYHKIRLFEKNTKKYEIVKYSIVPNEKLKQYFEDLGIDIKYLTGSVESGEKWLGENLGIDIKYLTVEQKSEVSEEKIKKFL Fusobacterium (SEQMGKPNRSSIIKIIISNYDNKGIKEVKVRYNKQAQLDTFLIKSELKDGKFI perfoetens ID NO:LYSIVDKAREKYRYSFEIDKTNINKNEILIIKKDIYSNKEDKVIRKYILSF ATCC 29250 125)EVSEKNDRTIVTKIKDCLETQKKEKFERENTRRLISETERKLLSEETQK T364DRAFT_TYSKIACCSPEDIDSVKIYKIKRYLAYRSNMLLFFSLINDIFVKGVVKD scaffold00009.9_NGEEVGEIWRIIDSKEIDEKKTYDLLVENFKKRMSQEFINYKQSIENKI CEKNTNKIKEIEQKLKKEKYKKEINRLKKQLIELNRENDLLEKDKIELSDEEIREDIEKILKIYSDLRHKLMHYNYQYFENLFENKKISKEKNEDVNLTELLDLNLFRYLPLVRQLKLENKTNYLEKEDKITVLGVSDSAIKYYSYYNFLCEQKNGFNNFINSFFSNDGEENKSFKEKINLSLEKEIEIMEKETNEKIKEINKNELQLMKEQKELGTAYVLDIHSLNDYKISHNERNKNVKLQNDIMNGNRDKNALDKINKKLVELKIKMDKITKRNSILRLKYKLQVAYGFLMEEYKGNIKKFKDEFDISKEKIKSYKSKGEKYLEVKSEKKYITKILNSIEDIHNITWLKNQEENNLFKFYVLTYILLPFEFRGDFLGFVKKHYYDIKNVEFLDENNDRLTPEQLEKMKNDSFFNKIRLFEKNSKKYDILKESILTSERIGKYFSLLNTGAKYFEYGGEENRGIFNKNIIIPIFKYYQIVLKLYNDVELAMLLTLSESDEKDINKIKELVTLKEKVSPKKIDYEKKYKFSVLLDCFNRIINLGKKDFLASEEVKEVAKTFTNLAYLRNKICHLNYSKFIDDLLTIDTNKSTTDSEGKLLINDRIRKLIKFIRENNQKMNISIDYNYINDYYMKKEKFIFGQRKQAKTIIDSGKKANKRNKAEELLKMYRVKKENINLIYELSKKLNELTKSELFLLDKKLLKDIDFTDVKIKNKSFFELKNDVKEVANIKQALQKHSSELIGIYKKEVIMAIKRSIVSKLIYDEEKVLSIIIYDKTNKKYEDFLLEIRRERDINKFQFLIDEKKEKLGYEKIIETKEKKKVVVKIQNNSELVSEPRIIKNKDKKKAKTPEEISKLGILDLTNHYCFNLKITL Fusobacterium (SEQMENKGNNKKIDFDENYNILVAQIKEYFTKEIENYNNRIDNIIDKKELLK ulcerans ATCC ID NO:YSEKKEESEKNKKLEELNKLKSQKLKILTDEEIKADVIKIIKIFSDLRHS 49185 cont2.38 126)LMHYEYKYFENLFENKKNEELAELLNLNLFKNLTLLRQMKIENKTNYLEGREEFNIIGKNIKAKEVLGHYNLLAEQKNGFNNFINSFFVQDGTENLEFKKLIDEHFVNAKKRLERNIKKSKKLEKELEKMEQHYQRLNCAYVWDIHTSTTYKKLYNKRKSLIEEYNKQINEIKDKEVITAINVELLRIKKEMEEITKSNSLFRLKYKMQIAYAFLEIEFGGNIAKFKDEFDCSKMEEVQKYLKKGVKYLKYYKDKEAQKNYEFPFEEIFENKDTHNEEWLENTSENNLFKFYILTYLLLPMEFKGDFLGVVKKHYYDIKNVDFTDESEKELSQVQLDKMIGDSFFHKIRLFEKNTKRYEIIKYSILTSDEIKRYFRLLELDVPYFEYEKGTDEIGIFNKNIILTIFKYYQIIFRLYNDLEIHGLFNISSDLDKILRDLKSYGNKNINFREFLYVIKQNNNSSTEEEYRKIWENLEAKYLRLHLLTPEKEEIKTKTKEELEKLNEISNLRNGICHLNYKEIIEEILKTEISEKNKEATLNEKIRKVINFIKENELDKVELGFNFINDFFMKKEQFMFGQIKQVKEGNSDSITTERERKEKNNKKLKETYELNCDNLSEFYETSNNLRERANSSSLLEDSAFLKKIGLYKVKNNKVNSKVKDEEKRIENIKRKLLKDSSDIMGMYKAEVVKKLKEKLILIFKHDEEKRIYVTVYDTSKAVPENISKEILVKRNNSKEEYFFEDNNKKYVTEYYTLEITETNELKVIPAKKLEGKEFK TEKNKENKLMLNNHYCFNVKIIYAnaerosalibacter (SEQ MKSGRREKAKSNKSSIVRVIISNFDDKQVKEIKVLYTKQGGIDVIKFKSsp. ND1 genome ID NO: TEKDEKGRMKFNFDCAYNRLEEEEFNSFGGKGKQSFFVTTNEDLTELassembly 127) HVTKRHKTTGEIIKDYTIQGKYTPIKQDRTKVTVSITDNKDHFDSNDLAnaerosalibacter GDKIRLSRSLTQYTNRILLDADVMKNYREIVCSDSEKVDETINIDSQEImassiliensis YKINRFLSYRSNMIIYYQMINNFLLHYDGEEDKGGNDSINLINEIWKYE ND1NKKNDEKEKIIERSYKSIEKSINQYILNHNTEVESGDKEKKIDISEERIKEDLKKTFILFSRLRHYMVHYNYKFYENLYSGKNFIIYNKDKSKSRRFSELLDLNIFKELSKIKLVKNRAVSNYLDKKTTIHVLNKNINAIKLLDIYRDICETKNGFNNFINNMMTISGEEDKEYKEMVTKHFNENMNKLSTYLENFKKHSDFKTNNKKKETYNLLKQELDEQKKLRLWFNAPYVYDIHSSKKYKELYVERKKYVDIHSKLIEAGINNDNKKKLNEINVKLCELNTEMKEMTKLNSKYRLQYKLQLAFGFILEEFNLDIDKFVSAFDKDNNLTISKFMEKRETYLSKSLDRRDNRFKKLIKDYKFRDTEDIFCSDRENNLVKLYILMYILLPVEIRGDFLGFVKKNYYDLKHVDFIDKRNNDNKDTFFHDLRLFEKNVKRLEVTSYSLSDGFLGKKSREKFGKELEKFIYKNVSIALPTNIDIKEFNKSLVLPMMKNYQIIFKLLNDIEISALFLIAKKEGNEGSITFKKVIDKVRKEDMNGNINFSQVMKMALNEKVNCQIRNSIAHINMKQLYIEPLNIYINNNQNKKTISEQMEEIIDICITKGLTGKELNKNIINDYYMKKEKLVFNLKLRKRNNLVSIDAQQKNMKEKSILNKYDLNYKDENLNIKEIILKVNDLNNKQKLLKETTEGESNYKNALSKDILLLNGIIRKNINFKIKEMILGIIQQNEYRYVNINIYDKIRKEDHNIDLKINNKYIEISCYENKSNESTDERINFKIKYMDLKVKNELLVPSCYEDIYIKKKIDLEIRYIENCKVVYIDIYYKKYNINLEFDGKTLFVKFNKDVKKNNQKVNLESNYIQNIKFIVS

The protein sequences of the C2c2 (Cas13a) species are listed in Table 8below.

TABLE 8 c2c2-5  1 Lachno-MQISKVNHKHVAVGQKDRERITGFIYNDPVGDEKSLEDVVAKRANDTKV spiraceaeLENVENTKDLYDSQESDKSEKDKEIISKGAKEVAKSENSAITILKKQNKIYS bacteriumTLTSQQVIKELKDKEGGARIYDDDIEEALTETLKKSFRKENVRNSIKVLIEN MA2020AAGIRSSLSKDEEELIQEYFVKQLVEEYTKTKLQKNVVKSIKNQNMVIQPD (SEQ IDSDSQVLSLSESRREKQSSAVSSDTLVNCKEKDVLKAFLTDYAVLDEDERNS NO: 128)LLWKLRNLVNLYFYGSESIRDYSYTKEKSVWKEHDEQKANKTLFIDEICHITKIGKNGKEQKVLDYEENRSRCRKQNINYYRSALNYAKNNTSGIFENEDSNHEWIHLIENEVERLYNGIENGEEFKFETGYISEKVWKAVINHLSIKYIALGKAVYNYAMKELSSPGDIEPGKIDDSYINGITSFDYEIIKAEESLQRDISMNVVFATNYLACATVDTDKDELLFSKEDIRSCTKKDGNLCKNIMQFWGGYSTWKNECEEYLKDDKDALELLYSLKSMLYSMRNSSFHESTENVDNGSWDTELIGKLFEEDCNRAARIEKEKEYNNNLHMFYSSSLLEKVLERLYSSHHERASQVPSENRVEVRKNEPSSLSEQRITPKFTDSKDEQIWQSAVYYLCKEIYYNDFLQSKEAYKLFREGVKNLDKNDINNQKAADSFKQAVVYYGKAIGNATLSQVCQAIMTEYNRQNNDGLKKKSAYAEKQNSNKYKHYPLELKQVLQSAFWEYLDENKEIYGFISAQIHKSNVEIKAEDFIANYSSQQYKKLVDKVKKTPELQKWYTLGRLINPRQANQFLGSIRNYVQFVKDIQRRAKENGNPIRNYYEVLESDSIIKILEMCTKLNGTTSNDIHDYFRDEDEYAEYISQFVNEGDVHSGAALNAFCNSESEGKKNGIYYDGINPIVNRNWVLCKLYGSPDLISKIISRVNENMIHDFFIKQEDLIREYQIKGICSNKKEQQDLRTFQVLKNRVELRDIVEYSEIINELYGQLIKWCYLRERDLMYFQLGEHYLCLNNASSKEADYIKINVDDRNISGAILYQIAAMYINGLPVYYKKDDMYVALKSGKKASDELNSNEQTSKKINYFLKYGNNILGDKKDQLYLAGLELFENVAEHENIIIERNEIDHEHYFYDRDRSMLDLYSEVEDREFTYDMKLRKNVVNMLYNILLDHNIVSSFVFETGEKKVGRGDSEVIKPSAKIRLRANNGVSSDVETYKVGSKDELKIATLPAKNEEFLLNVARLIYYPDMEAVSENMVREGVVKVEKSNDKKGKISRGSNTRSSNQSKYNNK SKNRMNYSMGSIFEKMDLKFDc2c2-6  2 Lachno- MKISKVREENRGAKLTVNAKTAVVSENRSQEGILYNDPSRYGKSRKNDEDspiraceae RDRYIESRLKSSGKLYRIFNEDKNKRETDELQWELSEIVKKINRRNGLVLS bacteriumDMLSVDDRAFEKAFEKYAELSYTNRRNKVSGSPAFETCGVDAATAERLKG NK4A179IISETNFINRIKNNIDNKVSEDIIDRIIAKYLKKSLCRERVKRGLKKLLMNAF (SEQ IDDLPYSDPDIDVQRDFIDYVLEDFYHVRAKSQVSRSIKNMNMPVQPEGDGK NO: 129)FAITVSKGGTESGNKRSAEKEAFKKELSDYASLDERVRDDMLRRMRRLVVLYFYGSDDSKLSDVNEKFDVWEDHAARRVDNREFIKLPLENKLANGKTDKDAERIRKNTVKELYRNQNIGCYRQAVKAVEEDNNGRYFDDKMLNMFFIHRIEYGVEKIYANLKQVTEFKARTGYLSEKIWKDLINYISIKYIAMGKAVYNYAMDELNASDKKEIELGKISEEYLSGISSFDYELIKAEEMLQRETAVYVAFAARHLSSQTVELDSENSDELLLKPKGTMDKNDKNKLASNNILNELKDKETLRDTILQYFGGHSLWTDFPFDKYLAGGKDDVDFLTDLKDVIYSMRNDSFHYATENHNNGKWNKELISAMFEHETERMTVVMKDKFYSNNLPMFYKNDDLKKLLIDLYKDNVERASQVPSENKVEVRKNEPALVRDKDNLGIELDLKADADKGENELKEYNALYYMEKEIYYNAFLNDKNVRERFITKATKVADNYDRNKERNLKDRIKSAGSDEKKKLREQLQNYIAENDFGQRIKNIVQVNPDYTLAQICQLIMTEYNQQNNGCMQKKSAARKDINKDSYQHYKMLLLVNLRKAFLEFIKENYAFVLKPYKHDLCDKADEVPDFAKYVKPYAGLISRVAGSSELQKWYIVSRELSPAQANHMLGELHSYKQYVWDIYRRASETGTEINHSIAEDKIAGVDITDVDAVIDLSVKLCGTISSEISDYFKDDEVYAEYISSYLDFEYDGGNYKDSLNRECNSDAVNDQKVALYYDGEHPKLNRNIILSKLYGERRFLEKITDRVSRSDIVEYYKLKKETSQYQTKGIFDSEDEQKNIKKFQEMKNIVEFRDLMDYSEIADELQGQLINWIYLRERDLMNFQLGYHYACLNNDSNKQATYVTLDYQGKKNRKINGAILYQICAMYINGLPLYYVDKDSSEWTVSDGKESTGAKIGEFYRYAKSFENTSDCYASGLEIFENISEHDNITELRNYIEHFRYYSSFDRSFLGIYSEVEDREFTYDLKYRKNVPTILYNILLQHFVNVRFEFVSGKKMIGIDKKDRKIAKEKECARITIREKNGVYSEQFTYKLKNGTVYVDARDKRYLQSIIRLLFYPEKVNMDEMIEVKEKKKPSDNNTGKGYSKRDRQQDRKEYDKYKEKKKKEGNFLSGMGGNINWDEINAQLKN c2c2-7  3 [Clostridium]MKESKVDHTRSAVGIQKATDSVHGMLYTDPKKQEVNDLDKREDQLNVK aminophilumAKRLYNVENQSKAEEDDDEKREGKVVKKLNRELKDLLEHREVSRYNSIGN DSMAKYNYYGIKSNPEEIVSNLGMVESLKGERDPQKVISKLLLYYLRKGLKPGT 10710DGLRMILEASCGLRKLSGDEKELKVFLQTLDEDFEKKTFKKNLIRSIENQN (SEQ IDMAVQPSNEGDPIIGITQGRENSQKNEEKSAIERMMSMYADLNEDHREDVL NO: 130)RKLRRLNVLYFNVDTEKTEEPTLPGEVDTNPVFEVWHDHEKGKENDRQFATFAKILTEDRETRKKEKLAVKEALNDLKSAIRDHNIMAYRCSIKVTEQDKDGLFFEDQRINREWIHHIESAVERILASINPEKLYKLRIGYLGEKVWKDLLNYLSIKYIAVGKAVEHFAMEDLGKTGQDIELGKLSNSVSGGLTSFDYEQIRADETLQRQLSVEVAFAANNLFRAVVGQTGKKIEQSKSEENEEDELLWKAEKIAESIKKEGEGNTLKSILQFFGGASSWDLNHFCAAYGNESSALGYETKFADDLRKAIYSLRNETFHFTTLNKGSFDWNAKLIGDMFSHEAATGIAVERTRFYSNNLPMFYRESDLKRIMDHLYNTYHPRASQVPSFNSVFVRKNFRLFLSNTLNTNTSFDTEVYQKWESGVYYLFKEIYYNSFLPSGDAHHLFFEGLRRIRKEADNLPIVGKEAKKRNAVQDFGRRCDELKNLSLSAICQMIMTEYNEQNNGNRKVKSTREDKRKPDIFQHYKMLLLRTLQEAFAIYIRREEFKFIFDLPKTLYVMKPVEEFLPNWKSGMFDSLVERVKQSPDLQRWYVLCKFLNGRLLNQLSGVIRSYIQFAGDIQRRAKANHNRLYMDNTQRVEYYSNVLEVVDFCIKGTSRFSNVFSDYFRDEDAYADYLDNYLQFKDEKIAEVSSFAALKTFCNEEEVKAGIYMDGENPVMQRNIVMAKLFGPDEVLKNVVPKVTREEIEEYYQLEKQIAPYRQNGYCKSEEDQKKLLRFQRIKNRVEFQTITEFSEIINELLGQLISWSFLRERDLLYFQLGFHYLCLHNDTEKPAEYKEISREDGTVIRNAILHQVAAMYVGGLPVYTLADKKLAAFEKGEADCKLSISKDTAGAGKKIKDFFRYSKYVLIKDRMLTDQNQKYTIYLAGLELFENTDEHDNITDVRKYVDHFKYYATSDENAMSILDLYSEIHDRFFTYDMKYQKNVANMLENILLRHFVLIRPEFFTGSKKVGEGKKITCKARAQIEIAENGMRSEDFTYKLSDGKKNISTCMIAARDQKYLNTVARLLYYPHEAKKSIVDTREKKNNKKTNRGDGTFNKQKGTARKEKDNGPREFNDTGFSNTPFAGFDPFRNS c2c2-8  5 CarnobacteriumMRITKVKIKLDNKLYQVTMQKEEKYGTLKLNEESRKSTAEILRLKKASFN gallinarumKSFHSKTINSQKENKNATIKKNGDYISQIFEKLVGVDTNKNIRKPKMSLTD DSM 4847LKDLPKKDLALFIKRKFKNDDIVEIKNLDLISLFYNALQKVPGEHFTDESW (SEQ IDADFCQEMMPYREYKNKFIERKIILLANSIEQNKGFSINPETFSKRKRVLHQ NO: 131)WAIEVQERGDFSILDEKLSKLAEIYNFKKMCKRVQDELNDLEKSMKKGKNPEKEKEAYKKQKNFKIKTIWKDYPYKTHIGLIEKIKENEELNQFNIEIGKYFEHYFPIKKERCTEDEPYYLNSETIATTVNYQLKNALISYLMQIGKYKQFGLENQVLDSKKLQEIGIYEGFQTKFMDACVFATSSLKNIIEPMRSGDILGKREFKEAIATSSFVNYHHFFPYFPFELKGMKDRESELIPFGEQTEAKQMQNIWALRGSVQQIRNEIFHSFDKNQKFNLPQLDKSNFEFDASENSTGKSQSYIETDYKFLFEAEKNQLEQFFIERIKSSGALEYYPLKSLEKLFAKKEMKFSLGSQVVAFAPSYKKLVKKGHSYQTATEGTANYLGLSYYNRYELKEESFQAQYYLLKLIYQYVFLPNFSQGNSPAFRETVKAILRINKDEARKKMKKNKKFLRKYAFEQVREMEFKETPDQYMSYLQSEMREEKVRKAEKNDKGFEKNITMNFEKLLMQIFVKGFDVFLTTFAGKELLLSSEEKVIKETEISLSKKINEREKTLKASIQVEHQLVATNSAISYWLFCKLLDSRHLNELRNEMIKFKQSRIKFNHTQHAELIQNLLPIVELTILSNDYDEKNDSQNVDVSAYFEDKSLYETAPYVQTDDRTRVSFRPILKLEKYHTKSLIEALLKDNPQFRVAATDIQEWMHKREEIGELVEKRKNLHTEWAEGQQTLGAEKREEYRDYCKKIDRFNWKANKVTLTYLSQLHYLITDLLGRMVGFSALFERDLVYFSRSFSELGGETYHISDYKNLSGVLRLNAEVKPIKIKNIKVIDNEENPYKGNEPEVKPFLDRLHAYLENVIGIKAVHGKIRNQTAHLSVLQLELSMIESMNNLRDLMAYDRKLKNAVTKSMIKILDKHGMILKLKIDENHKNFEIESLIPKEIIHLKDKAIKTNQVSEEYCQLVLALLTTNP GNQLN c2c2-9  6Carnobacterium MRMTKVKINGSPVSMNRSKLNGHLVWNGTTNTVNILTKKEQSFAASFLNgallinarum KTLVKADQVKGYKVLAENIFIIFEQLEKSNSEKPSVYLNNIRRLKEAGLKRF DSM 4847FKSKYHEEIKYTSEKNQSVPTKLNLIPLFFNAVDRIQEDKFDEKNWSYFCK (SEQ IDEMSPYLDYKKSYLNRKKEILANSIQQNRGFSMPTAEEPNLLSKRKQLFQQ NO: 132)WAMKFQESPLIQQNNFAVEQFNKEFANKINELAAVYNVDELCTAITEKLMNFDKDKSNKTRNFEIKKLWKQHPHNKDKALIKLFNQEGNEALNQFNIELGKYFEHYFPKTGKKESAESYYLNPQTIIKTVGYQLRNAFVQYLLQVGKLHQYNKGVLDSQTLQEIGMYEGFQTKFMDACVFASSSLRNIIQATTNEDILTREKFKKELEKNVELKHDLFFKTEIVEERDENPAKKIAMTPNELDLWAIRGAVQRVRNQIFHQQINKRHEPNQLKVGSFENGDLGNVSYQKTIYQKLFDAEIKDIEIYFAEKIKSSGALEQYSMKDLEKLFSNKELTLSLGGQVVAFAPSYKKLYKQGYFYQNEKTIELEQFTDYDFSNDVFKANYYLIKLIYHYVFLPQFSQANNKLFKDTVHYVIQQNKELNTTEKDKKNNKKIRKYAFEQVKLMKNESPEKYMQYLQREMQEERTIKEAKKTNEEKPNYNFEKLLIQIFIKGFDTFLRNFDLNLNPAEELVGTVKEKAEGLRKRKERIAKILNVDEQIKTGDEEIAFWIFAKLLDARHLSELRNEMIKFKQSSVKKGLIKNGDLIEQMQPILELCILSNDSESMEKESFDKIEVFLEKVELAKNEPYMQEDKLTPVKFRFMKQLEKYQTRNFIENLVIENPEFKVSEKIVLNWHEEKEKIADLVDKRTKLHEEWASKAREIEEYNEKIKKNKSKKLDKPAEFAKFAEYKIICEAIENFNRLDHKVRLTYLKNLHYLMIDLMGRMVGFSVLFERDFVYMGRSYSALKKQSIYLNDYDTFANIRDWEVNENKHLFGTSSSDLTFQETAEFKNLKKPMENQLKALLGVTNHSFEIRNNIAHLHVLRNDGKGEGVSLLSCMNDLRKLMSYDRKLKNAVTKAIIKILDKHGMILKLTNNDHTKPFEIESLKPKKIIHLEKSNHSFPMDQVSQEYCDLVKKML VFTN c2c2-  7Paludibacter MRVSKVKVKDGGKDKMVLVHRKTTGAQLVYSGQPVSNETSNILPEKKRQ 10propionicigenes SFDLSTLNKTIIKFDTAKKQKLNVDQYKIVEKIFKYPKQELPKQIKAEEILP WB4FLNHKFQEPVKYWKNGKEESFNLTLLIVEAVQAQDKRKLQPYYDWKTW (SEQ IDYIQTKSDLLKKSIENNRIDLTENLSKRKKALLAWETEFTASGSIDLTHYHK NO: 133)VYMTDVLCKMLQDVKPLTDDKGKINTNAYHRGLKKALQNHQPAIFGTREVPNEANRADNQLSIYHLEVVKYLEHYFPIKTSKRRNTADDIAHYLKAQTLKTTIEKQLVNAIRANIIQQGKTNHHELKADTTSNDLIRIKTNEAFVLNLTGTCAFAANNIRNMVDNEQTNDILGKGDFIKSLLKDNTNSQLYSFFFGEGLSTNKAEKETQLWGIRGAVQQIRNNVNHYKKDALKTVFNISNFENPTITDPKQQTNYADTIYKARFINELEKIPEAFAQQLKTGGAVSYYTIENLKSLLTTFQFSLCRSTIPFAPGFKKVFNGGINYQNAKQDESFYELMLEQYLRKENFAEESYNARYFMLKLIYNNLFLPGFTTDRKAFADSVGFVQMQNKKQAEKVNPRKKEAYAFEAVRPMTAADSIADYMAYVQSELMQEQNKKEEKVAEETRINFEKFVLQVFIKGFDSFLRAKEFDFVQMPQPQLTATASNQQKADKLNQLEASITADCKLTPQYAKADDATHIAFYVFCKLLDAAHLSNLRNELIKFRESVNEFKFHHLLEIIEICLLSADVVPTDYRDLYSSEADCLARLRPFIEQGADITNWSDLFVQSDKHSPVIHANIELSVKYGTTKLLEQIINKDTQFKTTEANFTAWNTAQKSIEQLIKQREDHHEQWVKAKNADDKEKQERKREKSNFAQKFIEKHGDDYLDICDYINTYNWLDNKMHFVHLNRLHGLTIELLGRMAGFVALFDRDFQFFDEQQIADEFKLHGFVNLHSIDKKLNEVPTKKIKEIYDIRNKIIQINGNKINESVRANLIQFISSKRNYYNNAFLHVSNDEIKEKQMYDIRNHIAHFNYLTKDAADFSLIDLINELRELLHYDRKLKNAVSKAFIDLFDKHGMILKLKLNADHKLKVESLEPKKIYHLGSSAKDKPEYQYCTNQVMMAYCNMCRSLLEMKK c2c2-  9 ListeriaMLALLHQEVPSQKLHNLKSLNTESLTKLEKPKFQNMISYPPSKGAEHVQF 11 weihenstephan-CLTDIAVPAIRDLDEIKPDWGIFFEKLKPYTDWAESYIHYKQTTIQKSIEQN ensisKIQSPDSPRKLVLQKYVTAFLNGEPLGLDLVAKKYKLADLAESEKVVDLNE FSL R9-DKSANYKIKACLQQHQRNILDELKEDPELNQYGIEVKKYIQRYFPIKRAPN 0317 (SEQRSKHARADFLKKELIESTVEQQFKNAVYHYVLEQGKMEAYELTDPKTKDL ID NO:QDIRSGEAFSFKFINACAFASNNLKMILNPECEKDILGKGDFKKNLPNSTT 134)QSDVVKKMIPFFSDEIQNVNEDEAIWAIRGSIQQIRNEVYHCKKHSWKSILKIKGFEFEPNNMKYTDSDMQKLMDKDIAKIPDFIEEKLKSSGIIREYSHDKLQSIWEMKQGFSLLTTNAPFVPSFKRVYAKGHDYQTSKNRYYDLGLTTEDILEYGEEDFRARYFLTKLVYYQQEMPWFTADNNAFRDAANFVLRLNKNRQQDAKAFINIREVEEGEMPRDYMGYVQGQIAIHEDSTEDTPNHFEKFISQVFIKGEDSHMRSADLKFIKNPRNQGLEQSEIEEMSFDIKVEPSFLKNKDDYIAFWTFCKMLDARHLSELRNEMIKYDGHLTGEQEIIGLALLGVDSRENDWKQFFSSEREYEKIMKGYVGEELYQREPYRQSDGKTPILFRGVEQARKYGTETVIQRLFDASPEEKVSKCNITEWERQKETIEETIERRKELHNEWEKNPKKPQNNAFFKEYKECCDAIDAYNWHKNKTTLVYVNELHHLLIEILGRYVGYVAIADRDEQCMANQYFKHSGITERVEYWGDNRLKSIKKLDTFLKKEGLEVSEKNARNHIAHLNYLSLKSECTLLYLSERLREIFKYDRKLKNAVSKSLIDILDRHGMSVVFANLKENKHRLVIKSLEPKKLRHLGEKKIDNGYIETNQVSEEY CGIVKRLLEI c2c2- 10Listeriaceae MKITKMRVDGRTIVMERTSKEGQLGYEGIDGNKTTEIIFDKKKESFYKSIL 12bacterium NKTVRKPDEKEKNRRKQAINKAINKEITELMLAVLHQEVPSQKLHNLKSL FSL M6-NTESLTKLEKPKFQNMISYPPSKGAEHVQFCLTDIAVPAIRDLDEIKPDWG 0635 =IFFEKLKPYTDWAESYIHYKQTTIQKSIEQNKIQSPDSPRKLVLQKYVTAFL ListeriaNGEPLGLDLVAKKYKLADLAESFKLVDLNEDKSANYKIKACLQQHQRNIL newyorkensisDELKEDPELNQYGIEVKKYIQRYFPIKRAPNRSKHARADFLKKELIESTVE FSL M6-QQFKNAVYHYVLEQGKMEAYELTDPKTKDLQDIRSGEAFSFKFINACAFA 0635 (SEQSNNLKMILNPECEKDILGKGNEKKNLPNSTTRSDVVKKMIPFFSDELQNV ID NO:NEDEAIWAIRGSIQQIRNEVYHCKKHSWKSILKIKGFEFEPNNMKYADSD 135)MQKLMDKDIAKIPEFIEEKLKSSGVVREYRHDELQSIWEMKQGFSLLTTNAPFVPSFKRVYAKGHDYQTSKNRYYNLDLTTEDILEYGEEDFRARYFLTKLVYYQQEMPWFTADNNAFRDAANFVLRLNKNRQQDAKAFINIREVEEGEMPRDYMGYVQGQIAIHEDSIEDTPNHFEKFISQVFIKGFDRHMRSANLKFIKNPRNQGLEQSEIEEMSFDIKVEPSFLKNKDDYIAFWIFCKMLDARHLSELRNEMIKYDGHLTGEQEIIGLALLGVDSRENDWKQFFSSEREYEKIMKGYVVEELYQREPYRQSDGKTPILFRGVEQARKYGTETVIQRLFDANPEEKVSKCNLAEWERQKETIEETIKRRKELHNEWAKNPKKPQNNAFFKEYKECCDAIDAYNWHKNKTTLAYVNELHHLLIEILGRYVGYVAIADRDFQCMANQYFKHSGITERVEYWGDNRLKSIKKLDTFLKKEGLEVSEKNARNHIAHLNYLSLKSECTLLYLSERLREIFKYDRKLKNAVSKSLIDILDRHGMSVVFANLKENKHRLVIKSLEPKKLRHLGGKKIDGGYIETNQVSEEYCGIVKRLLEM c2c2- 12 LeptotrichiaMKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYIKNPDNA 13 wadeiSEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISE F0279YDLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENK (SEQ IDANYQKINENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKK NO: 136)EDIEKLFFLIENSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFWSVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQKFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIESNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKYTENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDFELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEUNDEDYKEYEKAIGNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEEIFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKKIEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE c2c2- 15 RhodobacterMQIGKVQGRTISEFGDPAGGLKRKISTDGKNRKELPAHLSSDPKALIGQWI 14 capsulatusSGIDKIYRKPDSRKSDGKAIHSPTPSKMQFDARDDLGEAFWKLVSEAGLA SB 1003QDSDYDQFKRRLHPYGDKFQPADSGAKLKFEADPPEPQAFHGRWYGAM (SEQ IDSKRGNDAKELAAALYEHLHVDEKRIDGQPKRNPKTDKFAPGLVVARALGI NO: 137)ESSVLPRGMARLARNWGEEEIQTYFVVDVAASVKEVAKAAVSAAQAFDPPRQVSGRSLSPKVGFALAEHLERVTGSKRCSFDPAAGPSVLALHDEVKKTYKRLCARGKNAARAFPADKTELLALMRHTHENRVRNQMVRMGRVSEYRGQQAGDLAQSHYWTSAGQTEIKESEIFVRLWVGAFALAGRSMKAWIDPMGKIVNTEKNDRDLTAAVNIRQVISNKEMVAEAMARRGIYFGETPELDRLGAEGNEGFVFALLRYLRGCRNQTFHLGARAGFLKEIRKELEKTRWGKAKEAEHVVLTDKTVAAIRAIIDNDAKALGARLLADLSGAFVAHYASKEHFSTLYSEIVKAVKDAPEVSSGLPRLKLLLKRADGVRGYVHGLRDTRKHAFATKLPPPPAPRELDDPATKARYIALLRLYDGPFRAYASGITGTALAGPAARAKEAATALAQSVNVTKAYSDVMEGRTSRLRPPNDGETLREYLSALTGETATEFRVQIGYESDSENARKQAEFIENYRRDMLAFMFEDYIRAKGFDWILKIEPGATAMTRAPVLPEPIDTRGQYEHWQAALYLVMHFVPASDVSNLLHQLRKWEALQGKYELVQDGDATDQADARREALDLVKRFRDVLVLFLKTGEARFEGRAAPFDLKPFRALFANPATFDRLFMATPTTARPAEDDPEGDGASEPELRVARTLRGLRQIARYNHMAVLSDLFAKHKVRDEEVARLAEIEDETQEKSQIVAAQELRTDLHDKVMKCHPKTISPEERQSYAAAIKTIEEHRFLVGRVYLGDHLRLHRLMMDVIGRLIDYAGAYERDTGTFLINASKQLGAGADWAVTIAGAANTDARTQTRKDLAHFNVLDRADGTPDLTALVNRAREMMAYDRKRKNAVPRSILDMLARLGLTLKWQMKDHLLQDATITQAAIKHLDKVRLTVGGPAAVTEARFSQDYLQMVAAVFNGSVQNPKPRRRDDGDAWHKPPKPATAQSQPDQKPPNKAPSAGSRLPPPQVGEVYEGVVVKVIDTGSLGFLAVEGVAGNIGLHISRLRRIREDAIIVGRRYRFRVEIYVPPKSNTSKLNAADLVRID c2c2- 16 RhodobacterMQIGKVQGRTISEFGDPAGGLKRKISTDGKNRKELPAHLSSDPKALIGQWI 15 capsulatusSGIDKIYRKPDSRKSDGKAIHSPTPSKMQFDARDDLGEAFWKLVSEAGLA R121 (SEQQDSDYDQFKRRLHPYGDKFQPADSGAKLKFEADPPEPQAFHGRWYGAM ID NO:SKRGNDAKELAAALYEHLHVDEKRIDGQPKRNPKTDKFAPGLVVARALGI 138)ESSVLPRGMARLARNWGEEEIQTYFVVDVAASVKEVAKAAVSAAQAFDPPRQVSGRSLSPKVGFALAEHLERVTGSKRCSFDPAAGPSVLALHDEVKKTYKRLCARGKNAARAFPADKTELLALMRHTHENRVRNQMVRMGRVSEYRGQQAGDLAQSHYWTSAGQTEIKESEIFVRLWVGAFALAGRSMKAWIDPMGKIVNTEKNDRDLTAAVNIRQVISNKEMVAEAMARRGIYFGETPELDRLGAEGNEGFVFALLRYLRGCRNQTFHLGARAGELKEIRKELEKTRWGKAKEAEHVVLTDKTVAAIRAIIDNDAKALGARLLADLSGAFVAHYASKEHESTLYSEIVKAVKDAPEVSSGLPRLKLLLKRADGVRGYVHGLRDTRKHAFATKLPPPPAPRELDDPATKARYIALLRLYDGPFRAYASGITGTALAGPAARAKEAATALAQSVNVTKAYSDVMEGRSSRLRPPNDGETLREYLSALTGETATEFRVQIGYESDSENARKQAEFIENYRRDMLAFMFEDYIRAKGEDWILKIEPGATAMTRAPVLPEPIDTRGQYEHWQAALYLVMHFVPASDVSNLLHQLRKWEALQGKYELVQDGDATDQADARREALDLVKRFRDVLVLFLKTGEARFEGRAAPFDLKPFRALFANPATFDRLFMATPTTARPAEDDPEGDGASEPELRVARTLRGLRQIARYNHMAVLSDLFAKHKVRDEEVARLAEIEDETQEKSQIVAAQELRTDLHDKVMKCHPKTISPEERQSYAAAIKTIEEHRELVGRVYLGDHLRLHRLMMDVIGRLIDYAGAYERDTGTFLINASKQLGAGADWAVTIAGAANTDARTQTRKDLAHFNVLDRADGTPDLTALVNRAREMMAYDRKRKNAVPRSILDMLARLGLTLKWQMKDHLLQDATITQAAIKHLDKVRLTVGGPAAVTEARFSQDYLQMVAAVENGSVQNPKPRRRDDGDAWHKPPKPATAQSQPDQKPPNKAPSAGSRLPPPQVGEVYEGVVVKVIDTGSLGFLAVEGVAGNIGLHISRLRRIREDAIIVGRRYRFRVEIYVPPKSNTSKLNAADLVRID c2c2- 17 RhodobacterMQIGKVQGRTISEFGDPAGGLKRKISTDGKNRKELPAHLSSDPKALIGQWI 16 capsulatusSGIDKIYRKPDSRKSDGKAIHSPTPSKMQFDARDDLGEAFWKLVSEAGLA DE442QDSDYDQFKRRLHPYGDKFQPADSGAKLKFEADPPEPQAFHGRWYGAM (SEQ IDSKRGNDAKELAAALYEHLHVDEKRIDGQPKRNPKTDKFAPGLVVARALGI NO: 139)ESSVLPRGMARLARNWGEEEIQTYFVVDVAASVKEVAKAAVSAAQAFDPPRQVSGRSLSPKVGFALAEHLERVTGSKRCSFDPAAGPSVLALHDEVKKTYKRLCARGKNAARAFPADKTELLALMRHTHENRVRNQMVRMGRVSEYRGQQAGDLAQSHYWTSAGQTEIKESEIFVRLWVGAFALAGRSMKAWIDPMGKIVNTEKNDRDLTAAVNIRQVISNKEMVAEAMARRGIYFGETPELDRLGAEGNEGFVFALLRYLRGCRNQTFHLGARAGFLKEIRKELEKTRWGKAKEAEHVVLTDKTVAAIRAIIDNDAKALGARLLADLSGAFVAHYASKEHFSTLYSEIVKAVKDAPEVSSGLPRLKLLLKRADGVRGYVHGLRDTRKHAFATKLPPPPAPRELDDPATKARYIALLRLYDGPFRAYASGITGTALAGPAARAKEAATALAQSVNVTKAYSDVMEGRSSRLRPPNDGETLREYLSALTGETATEFRVQIGYESDSENARKQAEFIENYRRDMLAFMFEDYIRAKGFDWILKIEPGATAMTRAPVLPEPIDTRGQYEHWQAALYLVMHFVPASDVSNLLHQLRKWEALQGKYELVQDGDATDQADARREALDLVKRFRDVLVLFLKTGEARFEGRAAPFDLKPFRALFANPATFDRLFMATPTTARPAEDDPEGDGASEPELRVARTLRGLRQIARYNHMAVLSDLFAKHKVRDEEVARLAEIEDETQEKSQIVAAQELRTDLHDKVMKCHPKTISPEERQSYAAAIKTIEEHRELVGRVYLGDHLRLHRLMMDVIGRLIDYAGAYERDTGTFLINASKQLGAGADWAVTIAGAANTDARTQTRKDLAHFNVLDRADGTPDLTALVNRAREMMAYDRKRKNAVPRSILDMLARLGLTLKWQMKDHLLQDATITQAAIKHLDKVRLTVGGPAAVTEARFSQDYLQMVAAVENGSVQNPKPRRRDDGDAWHKPPKPATAQSQPDQKPPNKAPSAGSRLPPPQVGEVYEGVVVKVIDTGSLGFLAVEGVAGNIGLHISRLRRIREDAIIVGRRYRFRVEIYVPPKSNTSKLNAADLVRID c2c2-2 (SEQ IDMGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKI NO: 140)DNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNEMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADEVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIEGIFKKHYKVNEDSKKESKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDEFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVEKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNREFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIENNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIEDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIENSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLELNIGNPNEDKIYGLISNDIKMADAKFLENIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGEGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVEKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL c2c2-3 L wadeiMKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYIKNPDNA (Lw2)SEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISE (SEQ IDYDLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENK NO: 141)ANYQKINENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLEFLIENSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFWSVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQKFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIESNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKYTENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDFELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEUNDEDYKEYEKAIGNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEEIFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKKIEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALEKRPAATKKAGQAKKKKGSYPYDVPD YAYPYDVPDYAYPYDVPDYA*c2c2-4 Listeria MWISIKTLIHHLGVLFFCDYMYNRREKKIIEVKTMRITKVEVDRKKVLISRseeligeri DKNGGKLVYENEMQDNTEQIMHHKKSSFYKSVVNKTICRPEQKQMKKLV (SEQ IDHGLLQENSQEKIKVSDVTKLNISNFLNHRFKKSLYYFPENSPDKSEEYRIEI NO: 142)NLSQLLEDSLKKQQGTFICWESFSKDMELYINWAENYISSKTKLIKKSIRNNRIQSTESRSGQLMDRYMKDILNKNKPFDIQSVSEKYQLEKLTSALKATFKEAKKNDKEINYKLKSTLQNHERQIIEELKENSELNQFNIEIRKHLETYFPIKKTNRKVGDIRNLEIGEIQKIVNHRLKNKIVQRILQEGKLASYEIESTVNSNSLQKIKIEEAFALKFINACLFASNNLRNMVYPVCKKDILMIGEFKNSFKEIKHKKFIRQWSQFFSQEITVDDIELASWGLRGAIAPIRNEIIHLKKHSWKKFFNNPTFKVKKSKIINGKTKDVTSEFLYKETLFKDYFYSELDSVPELIINKMESSKILDYYSSDQLNQVFTIPNFELSLLTSAVPFAPSFKRVYLKGFDYQNQDEAQPDYNLKLNIYNEKAFNSEAFQAQYSLFKMVYYQVFLPQFTTNNDLFKSSVDFILTLNKERKGYAKAFQDIRKMNKDEKPSEYMSYIQSQLMLYQKKQEEKEKINHFEKFINQVFIKGFNSFIEKNRLTYICHPTKNTVPENDNIEIPFHTDMDDSNIAFWLMCKLLDAKQLSELRNEMIKFSCSLQSTEEISTFTKAREVIGLALLNGEKGCNDWKELFDDKEAWKKNMSLYVSEELLQSLPYTQEDGQTPVINRSIDLVKKYGTETILEKLFSSSDDYKVSAKDIAKLHEYDVTEKIAQQESLHKQWIEKPGLARDSAWTKKYQNVINDISNYQWAKTKVELTQVRHLHQLTIDLLSRLAGYMSIADRDFQFSSNYILERENSEYRVTSWILLSENKNKNKYNDYELYNLKNASIKVSSKNDPQLKVDLKQLRLTLEYLELFDNRLKEKRNNISHFNYLNGQLGNSILELFDDARDVLSYDRKLKNAVSKSLKEILSSHGMEVTFKPLYQTNHHLKIDKLQPKKIHHLGEKSTVSSNQVSNEYCQLVRTLLTM K

In certain embodiments, Cas13b is from an organism selected fromBergeyella, Prevotella, Porphyromonas, Bacteroides, Alistipes,Riemerella, Capnocytophaga, Flavobacterium, Myroides, Chryseobacterium,Paludibacter, Psychroflexus, Phaeodactylibacter Sinomicrobium, andReichenbachiella.

In certain embodiments, Cas13b is from an organism selected fromBergeyella zoohelcum, Prevotella intermedia, Prevotella buccae,Porphyromonas gingivalis, Bacteroides pyogenes, Alistipes sp. ZOR0009,Prevotella sp. MA2016, Prevotella sp. MA2016, Riemerella anatipestifer,Prevotella aurantiaca, Prevotella saccharolytica, HMPREF9712_03108[Myroides odoratimimus CCUG 10230], Prevotella intermedia,Capnocytophaga canimorsus, Porphyromonas gulae, Prevotella sp. P5-125,Flavobacterium branchiophilum, Myroides odoratimimus, Flavobacteriumcolumnare, Porphyromonas gingivalis, Porphyromonas sp. COT-052 OH4946,Prevotella intermedia, PIN17_0200 [Prevotella intermedia 17], Prevotellaintermedia, HMPREF6485_0083 [Prevotella buccae ATCC 33574],HMPREF9144_1146 [Prevotella pallens ATCC 700821], HMPREF9714_02132[Myroides odoratimimus CCUG 12901], HMPREF9711_00870 [Myroidesodoratimimus CCUG 3837], HMPREF9699_02005 [Bergeyella zoohelcum ATCC43767], HMPREF9151_01387 [Prevotella saccharolytica F0055], A343_1752[Porphyromonas gingivalis JCVI SC001], HMPREF1981_03090 [Bacteroidespyogenes F0041], HMPREF1553_02065 [Porphyromonas gingivalis F0568],HMPREF1988_01768 [Porphyromonas gingivalis F0185], HMPREF1990_01800[Porphyromonas gingivalis W4087], M573_117042 [Prevotella intermediaZT], A2033_10205 [Bacteroidetes bacterium GWA2_31_9], SAMN05421542_0666[Chryseobacterium jejuense], SAMN05444360_11366 [Chryseobacteriumcarnipullorum], SAMN05421786_1011119 [Chryseobacterium ureilyticum],Prevotella buccae, Porphyromonas gingivalis, Porphyromonas gingivalis,Prevotella pallens, Myroides odoratimimus, Myroides odoratimimus,Prevotella sp. MSX73, Porphyromonas gingivalis, Paludibacterpropionicigenes, Porphyromonas gingivalis, Flavobacterium columnare,Psychroflexus torquis, Riemerella anatipestifer, Prevotella pleuritidis,Porphyromonas gingivalis, Porphyromonas gingivalis, Porphyromonasgingivalis, Porphyromonas gingivalis, Porphyromonas gingivalis,Prevotella falsenii, Prevotella pleuritidis, [Porphyromonas gingivalis,Porphyromonas gulae, Porphyromonas gulae, Porphyromonas gulae,Porphyromonas gulae, Porphyromonas gulae, Porphyromonas gulae,Porphyromonas gulae, Capnocytophaga cynodegmi, Prevotella sp. P5-119,Prevotella sp. P4-76, Prevotella sp. P5-60, Phaeodactylibacterxiamenensis, Flavobacterium sp. 316, Porphyromonas gulae, WP_047431796,Riemerella anatipestifer, Porphyromonas gingivalis, Porphyromonasgingivalis, Flavobacterium columnare, Porphyromonas gingivalis,Porphyromonas gingivalis, Riemerella anatipestifer, Flavobacteriumcolumnare, Riemerella anatipestifer, Sinomicrobium oceani, andReichenbachiella agariperforans.

In certain embodiments, the effector protein may be a Listeria sp.C2c2p, preferably Listeria seeligeria C2c2p, more preferably Listeriaseeligeria serovar 1/2b str. SLCC3954 C2c2p and the crRNA sequence maybe 44 to 47 nucleotides in length, with a 5′ 29-nt direct repeat (DR)and a 15-nt to 18-nt spacer.

In certain embodiments, the effector protein may be a Leptotrichia sp.C2c2p, preferably Leptotrichia shahii C2c2p, more preferablyLeptotrichia shahii DSM 19757 C2c2p and the crRNA sequence may be 42 to58 nucleotides in length, with a 5′direct repeat of at least 24 nt, suchas a 5′ 24-28-nt direct repeat (DR) and a spacer of at least 14 nt, suchas a 14-nt to 28-nt spacer, or a spacer of at least 18 nt, such as 19,20, 21, 22, or more nt, such as 18-28, 19-28, 20-28, 21-28, or 22-28 nt.

More preferably, the effector protein may be a Leptotrichia sp.,preferably Leptotrichia wadei F0279, or a Listeria sp., preferablyListeria newyorkensis FSL M6-0635.

In certain embodiments, the effector protein may be a Type VI locieffector protein, more particularly a C2c2 or Cas13b, and the crRNAsequence may be 36 to 63 nucleotides in length, preferably 37-nt to62-nt in length, or 38-nt to 61-nt in length, or 39-nt to 60-nt inlength, more preferably 40-nt to 59-nt in length, or 41-nt to 58-nt inlength, most preferably 42-nt to 57-nt in length. For example, the crRNAmay comprise, consist essentially of or consist of a direct repeat (DR),preferably a 5′ DR, 26-nt to 31-nt in length, preferably 27-nt to 30-ntin length, even more preferably 28-nt or 29-nt in length or at least 28or 29 nt in length, and a spacer 10-nt to 32-nt in length, preferably11-nt to 31-nt in length, more preferably 12-nt to 30-nt in length, evenmore preferably 13-nt to 29-nt in length, and most preferably 14-nt to28-nt in length, such as 18-28 nt, 19-28 nt, 20-28 nt, 21-28 nt, or22-28 nt.

In certain example embodiments, the RNA-targeting effector protein is aCas13c effector protein as disclosed in U.S. Provisional PatentApplication No. 62/525,165 filed Jun. 26, 2017, and PCT Application No.US 2017/047193 filed Aug. 16, 2017. Example wildtype orthologuesequences of Cas13c are provided in Table 9 below.

TABLE 9 Name EHO19081 (SEQ. ID. No. 124) WP_094899336 WP_040490876WP_047396607 WP_035935671 WP_035906563 WP_042678931 WP_062627846WP_005959231 WP_027128616 WP_062624740 WP_096402050

The application further provides orthologs of C2c2 which demonstraterobust activity making them particularly suitable for differentapplications of RNA cleavage and detection. These applications includebut are not limited to those described herein. More particularly, anortholog which is demonstrated to have stronger activity than otherstested is the C2c2 ortholog identified from the organism Leptotrichiawadei (LwC2c2). The application thus provides methods for modifying atarget locus of interest, comprising delivering to said locus anon-naturally occurring or engineered composition comprising a CRISPReffector protein, more particularly a CRISPR effector protein withincreased activity as described herein and one or more nucleic acidcomponents, wherein at least the one or more nucleic acid components isengineered, the one or more nucleic acid components directs the complexto the target of interest and the effector protein forms a complex withthe one or more nucleic acid components and the complex binds to thetarget locus of interest. In particular embodiments, the target locus ofinterest comprises RNA. The application further provides for the use ofthe C2c2 effector proteins with increased activity in RNA sequencespecific interference, RNA sequence specific gene regulation, screeningof RNA or RNA products or lincRNA or non-coding RNA, or nuclear RNA, ormRNA, mutagenesis, Fluorescence in situ hybridization, or breeding.

In some embodiments, the Cas sequence is fused to one or more nuclearlocalization sequences (NLSs) or nuclear export signals (NESs), such asabout or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs orNESs. In some embodiments, the Cas comprises about or more than about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs or NESs at or near theamino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more NLSs or NESs at or near the carboxy-terminus, or a combinationof these (e.g. zero or at least one or more NLS or NES at theamino-terminus and zero or at one or more NLS or NES at the carboxyterminus). When more than one NLS or NES is present, each may beselected independently of the others, such that a single NLS or NES maybe present in more than one copy and/or in combination with one or moreother NLSs or NESs present in one or more copies. In a preferredembodiment of the invention, the Cas comprises at most 6 NLSs. In someembodiments, an NLS or NES is considered near the N- or C-terminus whenthe nearest amino acid of the NLS or NES is within about 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptidechain from the N- or C-terminus. Non-limiting examples of NLSs includean NLS sequence derived from: the NLS of the SV40 virus large T-antigen,having the amino acid sequence PKKKRKV (SEQ ID NO: 143; the NLS fromnucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequenceKRPAATKKAGQAKKKK) (SEQ ID NO: 144); the c-myc NLS having the amino acidsequence PAAKRVKLD (SEQ ID NO: 145) or RQRRNELKRSP (SEQ ID NO: 146); thehRNPA1 M9 NLS having the sequenceNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 147); the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:148) of the IBBdomain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 149) andPPKKARED (SEQ ID NO: 150) of the myoma T protein; the sequence POPKKKPL(SEQ ID NO: 151) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO:152) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 153) and PKQKKRK(SEQ ID NO: 154) of the influenza virus NS 1; the sequence RKLKKKIKKL(SEQ ID NO: 155) of the Hepatitis virus delta antigen; the sequenceREKKKFLKRR (SEQ ID NO: 156) of the mouse Mxl protein; the sequenceKRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 157) of the human poly(ADP-ribose)polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 158) of thesteroid hormone receptors (human) glucocorticoid. Non-limiting examplesof NESs include an NES sequence LYPERLRRILT (SEQ ID No, 159)(ctgtaccctgagcggctgcggcggatcctgacc (SEQ. ID No. 160). In general, theone or more NLSs or NESs are of sufficient strength to driveaccumulation of the Cas in a detectable amount in respectively thenucleus or the cytoplasm of a eukaryotic cell. In general, strength ofnuclear localization/export activity may derive from the number ofNLSs/NESs in the Cas, the particular NLS(s) or NES(s) used, or acombination of these factors. Detection of accumulation in thenucleus/cytoplasm may be performed by any suitable technique. Forexample, a detectable marker may be fused to the Cas, such that locationwithin a cell may be visualized, such as in combination with a means fordetecting the location of the nucleus (e.g. a stain specific for thenucleus such as DAPI) or cytoplasm. Cell nuclei may also be isolatedfrom cells, the contents of which may then be analyzed by any suitableprocess for detecting protein, such as immunohistochemistry, Westernblot, or enzyme activity assay. Accumulation in the nucleus may also bedetermined indirectly, such as by an assay for the effect of CRISPRcomplex formation (e.g. assay for DNA cleavage or mutation at the targetsequence, or assay for altered gene expression activity affected byCRISPR complex formation and/or Cas enzyme activity), as compared to acontrol no exposed to the Cas or complex, or exposed to a Cas lackingthe one or more NLSs or NESs. In certain embodiments, other localizationtags may be fused to the Cas protein, such as without limitation forlocalizing the Cas to particular sites in a cell, such as organells,such mitochondria, plastids, chloroplast, vesicles, golgi, (nuclear orcellular) membranes, ribosomes, nucleoluse, ER, cytoskeleton, vacuoles,centrosome, nucleosome, granules, centrioles, etc.

According to one aspect the invention provides non-naturally occurringor engineered composition comprising a guide RNA comprising a guidesequence capable of hybridizing to a target sequence in a genomic locusof interest in a cell, wherein the guide RNA is modified by theinsertion of one or more distinct RNA sequence(s) that bind an adaptorprotein. In particular embodiments, the RNA sequences may bind to two ormore adaptor proteins (e.g. aptamers), and wherein each adaptor proteinis associated with one or more functional domains. The guide RNAs of theCRISPR enzymes described herein are shown to be amenable to modificationof the guide sequence. In particular embodiments, the guide RNA ismodified by the insertion of distinct RNA sequence(s) 5′ of the directrepeat, within the direct repeat, or 3′ of the guide sequence. Whenthere is more than one functional domain, the functional domains can besame or different, e.g., two of the same or two different activators orrepressors. In an aspect the invention provides a herein-discussedcomposition, wherein the one or more functional domains are attached tothe RNA targeting enzyme so that upon binding to the target RNA thefunctional domain is in a spatial orientation allowing for thefunctional domain to function in its attributed function; In an aspectthe invention provides a herein-discussed composition, wherein thecomposition comprises a CRISPR-Cas complex having at least threefunctional domains, at least one of which is associated with the RNAtargeting enzyme and at least two of which are associated with the gRNA.

Accordingly, In an aspect the invention provides non-naturally occurringor engineered CRISPR-Cas complex composition comprising the guide RNA asherein-discussed and a CRISPR enzyme which is an RNA targeting enzyme,wherein optionally the RNA targeting enzyme comprises at least onemutation, such that the RNA targeting enzyme has no more than 5% of thenuclease activity of the enzyme not having the at least one mutation,and optionally one or more comprising at least one or more nuclearlocalization sequences. In particular embodiments, the guide RNA isadditionally or alternatively modified so as to still ensure binding ofthe RNA targeting enzyme but to prevent cleavage by the RNA targetingenzyme (as detailed elsewhere herein).

In particular embodiments, the RNA targeting enzyme is a CRISPR enzymewhich has a diminished nuclease activity of at least 97%, or 100% ascompared with the CRISPR enzyme not having the at least one mutation. Inan aspect the invention provides a herein-discussed composition, whereinthe CRISPR enzyme comprises two or more mutations. The mutations may beselected from mutations of one or more of the following amino acidresidues: R597, H602, R1278, and H1283, such as for instance one or moreof the following mutations: R597A, H602A, R1278A, and H1283A, accordingto Leptotrichia shahii CRISPR protein or a corresponding position in anortholog.

In particular embodiments, an RNA targeting system is provided asdescribed herein above comprising two or more functional domains. Inparticular embodiments, the two or more functional domains areheterologous functional domain. In particular embodiments, the systemcomprises an adaptor protein which is a fusion protein comprising afunctional domain, the fusion protein optionally comprising a linkerbetween the adaptor protein and the functional domain. In particularembodiments, the linker includes a GlySer linker. Additionally oralternatively, one or more functional domains are attached to the RNAeffector protein by way of a linker, optionally a GlySer linker. Inparticular embodiments, the one or more functional domains are attachedto the RNA targeting enzyme through one or both of the HEPN domains.

In an aspect the invention provides a herein-discussed composition,wherein the one or more functional domains associated with the adaptorprotein or the RNA targeting enzume is a domain capable of activating orrepressing RNA translation. In an aspect the invention provides aherein-discussed composition, wherein at least one of the one or morefunctional domains associated with the adaptor protein have one or moreactivities comprising methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,DNA integration activity RNA cleavage activity, DNA cleavage activity ornucleic acid binding activity, or molecular switch activity or chemicalinducibility or light inducibility.

In an aspect the invention provides a herein-discussed compositioncomprising an aptamer sequence. In particular embodiments, the aptamersequence is two or more aptamer sequences specific to the same adaptorprotein. In an aspect the invention provides a herein-discussedcomposition, wherein the aptamer sequence is two or more aptamersequences specific to different adaptor protein. In an aspect theinvention provides a herein-discussed composition, wherein the adaptorprotein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34,JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5,ϕCb8r, ϕCb12r, ϕCb23r, 7s, PRR1. Accordingly, in particular embodiments,the aptamer is selected from a binding protein specifically binding anyone of the adaptor proteins listed above. In an aspect the inventionprovides a herein-discussed composition, wherein the cell is aeukaryotic cell. In an aspect the invention provides a herein-discussedcomposition, wherein the eukaryotic cell is a mammalian cell, a plantcell or a yeast cell, whereby the mammalian cell is optionally a mousecell. In an aspect the invention provides a herein-discussedcomposition, wherein the mammalian cell is a human cell.

In an aspect the invention provides a herein above-discussed compositionwherein there is more than one gRNA, and the gRNAs target differentsequences whereby when the composition is employed, there ismultiplexing. In an aspect the invention provides a composition whereinthere is more than one gRNA modified by the insertion of distinct RNAsequence(s) that bind to one or more adaptor proteins.

In an aspect the invention provides a herein-discussed compositionwherein one or more adaptor proteins associated with one or morefunctional domains is present and bound to the distinct RNA sequence(s)inserted into the guide RNA(s).

In an aspect the invention provides a herein-discussed compositionwherein the guide RNA is modified to have at least one non-codingfunctional loop; e.g., wherein the at least one non-coding functionalloop is repressive; for instance, wherein at least one non-codingfunctional loop comprises Alu.

In an aspect the invention provides a nucleic acid molecule(s) encodingguide RNA or the RNA targeting CRISPR-Cas complex or the composition asherein-discussed. In an aspect the invention provides a vectorcomprising: a nucleic acid molecule encoding a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, wherein the direct repeat ofthe gRNA is modified by the insertion of distinct RNA sequence(s) thatbind(s) to two or more adaptor proteins, and wherein each adaptorprotein is associated with one or more functional domains; or, whereinthe gRNA is modified to have at least one non-coding functional loop. Inan aspect the invention provides vector(s) comprising nucleic acidmolecule(s) encoding: non-naturally occurring or engineered CRISPR-Cascomplex composition comprising the gRNA herein-discussed, and an RNAtargeting enzyme, wherein optionally the RNA targeting enzyme comprisesat least one mutation, such that the RNA targeting enzyme has no morethan 5% of the nuclease activity of the RNA targeting enzyme not havingthe at least one mutation, and optionally one or more comprising atleast one or more nuclear localization sequences. In an aspect a vectorcan further comprise regulatory element(s) operable in a eukaryotic celloperably linked to the nucleic acid molecule encoding the guide RNA(gRNA) and/or the nucleic acid molecule encoding the RNA targetingenzyme and/or the optional nuclear localization sequence(s).

The use of two different aptamers (each associated with a distinctnucleic acid-targeting guide RNAs) allows an activator-adaptor proteinfusion and a repressor-adaptor protein fusion to be used, with differentnucleic acid-targeting guide RNAs, to activate expression of one DNA orRNA, whilst repressing another. They, along with their different guideRNAs can be administered together, or substantially together, in amultiplexed approach. A large number of such modified nucleicacid-targeting guide RNAs can be used all at the same time, for example10 or 20 or 30 and so forth, whilst only one (or at least a minimalnumber) of effector protein molecules need to be delivered, as acomparatively small number of effector protein molecules can be usedwith a large number modified guides. The adaptor protein may beassociated (preferably linked or fused to) one or more activators or oneor more repressors. For example, the adaptor protein may be associatedwith a first activator and a second activator. The first and secondactivators may be the same, but they are preferably differentactivators. Three or more or even four or more activators (orrepressors) may be used, but package size may limit the number beinghigher than 5 different functional domains. Linkers are preferably used,over a direct fusion to the adaptor protein, where two or morefunctional domains are associated with the adaptor protein. Suitablelinkers might include the GlySer linker.

It is also envisaged that the nucleic acid-targeting effectorprotein-guide RNA complex as a whole may be associated with two or morefunctional domains. For example, there may be two or more functionaldomains associated with the nucleic acid-targeting effector protein, orthere may be two or more functional domains associated with the guideRNA (via one or more adaptor proteins), or there may be one or morefunctional domains associated with the nucleic acid-targeting effectorprotein and one or more functional domains associated with the guide RNA(via one or more adaptor proteins).

The fusion between the adaptor protein and the activator or repressormay include a linker. For example, GlySer linkers GGGS can be used. Theycan be used in repeats of 3 ((GGGGS)3) or 6, 9 or even 12 (SEQ ID Nos.161-164) or more, to provide suitable lengths, as required. Linkers canbe used between the guide RNAs and the functional domain (activator orrepressor), or between the nucleic acid-targeting effector protein andthe functional domain (activator or repressor). The linkers the user toengineer appropriate amounts of “mechanical flexibility”.

The invention comprehends a nucleic acid-targeting complex comprising anucleic acid-targeting effector protein and a guide RNA, wherein thenucleic acid-targeting effector protein comprises at least one mutation,such that the nucleic acid-targeting Cas protein has no more than 5% ofthe activity of the nucleic acid-targeting Cas protein not having the atleast one mutation and, optionally, at least one or more nuclearlocalization sequences; the guide RNA comprises a guide sequence capableof hybridizing to a target sequence in a RNA of interest in a cell; andwherein: the nucleic acid-targeting effector protein is associated withtwo or more functional domains; or at least one loop of the guide RNA ismodified by the insertion of distinct RNA sequence(s) that bind to oneor more adaptor proteins, and wherein the adaptor protein is associatedwith two or more functional domains; or the nucleic acid-targetingeffector protein is associated with one or more functional domains andat least one loop of the guide RNA is modified by the insertion ofdistinct RNA sequence(s) that bind to one or more adaptor proteins, andwherein the adaptor protein is associated with one or more functionaldomains.

In an aspect the invention provides a method for modifying geneexpression comprising the administration to a host or expression in ahost in vivo of one or more of the compositions as herein-discussed.

In an aspect the invention provides a herein-discussed method comprisingthe delivery of the composition or nucleic acid molecule(s) codingtherefor, wherein said nucleic acid molecule(s) are operatively linkedto regulatory sequence(s) and expressed in vivo. In an aspect theinvention provides a herein-discussed method wherein the expression invivo is via a lentivirus, an adenovirus, or an AAV.

Destabilized CRISPR Effector

In certain embodiments, the effecteor protein (CRISPR enzyme; e.g.Cas13a, Cas13b, or Cas 13c) according to the invention as describedherein is associated with or fused to a destabilization domain (DD). Insome embodiments, the DD is ER50. A corresponding stabilizing ligand forthis DD is, in some embodiments, 4HT. As such, in some embodiments, oneof the at least one DDs is ER50 and a stabilizing ligand therefor is4HT. or CMP8 In some embodiments, the DD is DHFR50. A correspondingstabilizing ligand for this DD is, in some embodiments, TMP. As such, insome embodiments, one of the at least one DDs is DHFR50 and astabilizing ligand therefor is TMP. In some embodiments, the DD is ER50.A corresponding stabilizing ligand for this DD is, in some embodiments,CMP8. CMP8 may therefore be an alternative stabilizing ligand to 4HT inthe ER50 system. While it may be possible that CMP8 and 4HT can/shouldbe used in a competitive matter, some cell types may be more susceptibleto one or the other of these two ligands, and from this disclosure andthe knowledge in the art the skilled person can use CMP8 and/or 4HT.

In some embodiments, one or two DDs may be fused to the N-terminal endof the CRISPR enzyme with one or two DDs fused to the C-terminal of theCRISPR enzyme. In some embodiments, the at least two DDs are associatedwith the CRISPR enzyme and the DDs are the same DD, i.e. the DDs arehomologous. Thus, both (or two or more) of the DDs could be ER50 DDs.This is preferred in some embodiments. Alternatively, both (or two ormore) of the DDs could be DHFR50 DDs. This is also preferred in someembodiments. In some embodiments, the at least two DDs are associatedwith the CRISPR enzyme and the DDs are different DDs, i.e. the DDs areheterologous. Thus, one of the DDS could be ER50 while one or more ofthe DDs or any other DDs could be DHFR50. Having two or more DDs whichare heterologous may be advantageous as it would provide a greater levelof degradation control. A tandem fusion of more than one DD at the N orC-term may enhance degradation; and such a tandem fusion can be, forexample ER50-ER50-C2c2 or DHFR-DHFR-C2c2 It is envisaged that highlevels of degradation would occur in the absence of either stabilizingligand, intermediate levels of degradation would occur in the absence ofone stabilizing ligand and the presence of the other (or another)stabilizing ligand, while low levels of degradation would occur in thepresence of both (or two of more) of the stabilizing ligands. Controlmay also be imparted by having an N-terminal ER50 DD and a C-terminalDHFR50 DD.

In some embodiments, the fusion of the CRISPR enzyme with the DDcomprises a linker between the DD and the CRISPR enzyme. In someembodiments, the linker is a GlySer linker. In some embodiments, theDD-CRISPR enzyme further comprises at least one Nuclear Export Signal(NES). In some embodiments, the DD-CRISPR enzyme comprises two or moreNESs. In some embodiments, the DD-CRISPR enzyme comprises at least oneNuclear Localization Signal (NLS). This may be in addition to an NES. Insome embodiments, the CRISPR enzyme comprises or consists essentially ofor consists of a localization (nuclear import or export) signal as, oras part of, the linker between the CRISPR enzyme and the DD. HA or Flagtags are also within the ambit of the invention as linkers. Applicantsuse NLS and/or NES as linker and also use Glycine Serine linkers asshort as GS up to (GGGGS)3.

Destabilizing domains have general utility to confer instability to awide range of proteins; see, e.g., Miyazaki, J Am Chem Soc. Mar. 7,2012; 134(9): 3942-3945, incorporated herein by reference. CMP8 or4-hydroxytamoxifen can be destabilizing domains. More generally, Atemperature-sensitive mutant of mammalian DHFR (DHFRts), a destabilizingresidue by the N-end rule, was found to be stable at a permissivetemperature but unstable at 37° C. The addition of methotrexate, ahigh-affinity ligand for mammalian DHFR, to cells expressing DHFRtsinhibited degradation of the protein partially. This was an importantdemonstration that a small molecule ligand can stabilize a proteinotherwise targeted for degradation in cells. A rapamycin derivative wasused to stabilize an unstable mutant of the FRB domain of mTOR (FRB*)and restore the function of the fused kinase, GSK-3β.6,7 This systemdemonstrated that ligand-dependent stability represented an attractivestrategy to regulate the function of a specific protein in a complexbiological environment. A system to control protein activity can involvethe DD becoming functional when the ubiquitin complementation occurs byrapamycin induced dimerization of FK506-binding protein and FKBP12.Mutants of human FKBP12 or ecDHFR protein can be engineered to bemetabolically unstable in the absence of their high-affinity ligands,Shield-1 or trimethoprim (TMP), respectively. These mutants are some ofthe possible destabilizing domains (DDs) useful in the practice of theinvention and instability of a DD as a fusion with a CRISPR enzymeconfers to the CRISPR protein degradation of the entire fusion proteinby the proteasome. Shield-1 and TMP bind to and stabilize the DD in adose-dependent manner. The estrogen receptor ligand binding domain(ERLBD, residues 305-549 of ERS1) can also be engineered as adestabilizing domain. Since the estrogen receptor signaling pathway isinvolved in a variety of diseases such as breast cancer, the pathway hasbeen widely studied and numerous agonist and antagonists of estrogenreceptor have been developed. Thus, compatible pairs of ERLBD and drugsare known. There are ligands that bind to mutant but not wild-type formsof the ERLBD. By using one of these mutant domains encoding threemutations (L384M, M421G, G521R)12, it is possible to regulate thestability of an ERLBD-derived DD using a ligand that does not perturbendogenous estrogen-sensitive networks. An additional mutation (Y537S)can be introduced to further destabilize the ERLBD and to configure itas a potential DD candidate. This tetra-mutant is an advantageous DDdevelopment. The mutant ERLBD can be fused to a CRISPR enzyme and itsstability can be regulated or perturbed using a ligand, whereby theCRISPR enzyme has a DD. Another DD can be a 12-kDa (107-amino-acid) tagbased on a mutated FKBP protein, stabilized by Shield1 ligand; see,e.g., Nature Methods 5, (2008). For instance a DD can be a modifiedFK506 binding protein 12 (FKBP12) that binds to and is reversiblystabilized by a synthetic, biologically inert small molecule, Shield-1;see, e.g., Banaszynski L A, Chen L C, Maynard-Smith L A, Ooi A G,Wandless T J. A rapid, reversible, and tunable method to regulateprotein function in living cells using synthetic small molecules. Cell.2006; 126:995-1004; Banaszynski L A, Sellmyer M A, Contag C H, WandlessT J, Thorne S H. Chemical control of protein stability and function inliving mice. Nat Med. 2008; 14:1123-1127; Maynard-Smith L A, Chen L C,Banaszynski L A, Ooi A G, Wandless T J. A directed approach forengineering conditional protein stability using biologically silentsmall molecules. The Journal of biological chemistry. 2007;282:24866-24872; and Rodriguez, Chem Biol. Mar. 23, 2012; 19(3):391-398-all of which are incorporated herein by reference and may beemployed in the practice of the invention in selected a DD to associatewith a CRISPR enzyme in the practice of this invention. As can be seen,the knowledge in the art includes a number of DDs, and the DD can beassociated with, e.g., fused to, advantageously with a linker, to aCRISPR enzyme, whereby the DD can be stabilized in the presence of aligand and when there is the absence thereof the DD can becomedestabilized, whereby the CRISPR enzyme is entirely destabilized, or theDD can be stabilized in the absence of a ligand and when the ligand ispresent the DD can become destabilized; the DD allows the CRISPR enzymeand hence the CRISPR-Cas complex or system to be regulated orcontrolled-turned on or off so to speak, to thereby provide means forregulation or control of the system, e.g., in an in vivo or in vitroenvironment. For instance, when a protein of interest is expressed as afusion with the DD tag, it is destabilized and rapidly degraded in thecell, e.g., by proteasomes. Thus, absence of stabilizing ligand leads toa D associated Cas being degraded. When a new DD is fused to a proteinof interest, its instability is conferred to the protein of interest,resulting in the rapid degradation of the entire fusion protein. Peakactivity for Cas is sometimes beneficial to reduce off-target effects.Thus, short bursts of high activity are preferred. The present inventionis able to provide such peaks. In some senses the system is inducible.In some other senses, the system repressed in the absence of stabilizingligand and de-repressed in the presence of stabilizing ligand.

In certain embodiments, the activity of the CRISPR effector depends onthe presence of two HEPN domains. These have been shown to be RNasedomains, i.e. nuclease (in particular an endonuclease) cutting RNA. C2c2HEPN may also target DNA, or potentially DNA and/or RNA. On the basisthat that the HEPN domains of C2c2 are at least capable of binding toand, in their wild-type form, cutting RNA, then it is preferred that theCRISPR effector, such as C2c2 or Cas13b effector protein has RNasefunction. It may also, or alternatively, have DNase function.

Thus, in some embodiments, the effector protein may be a RNA-bindingprotein, such as a dead-Cas type effector protein, which may beoptionally functionalised as described herein for instance with antranscriptional activator or repressor domain, NLS or other functionaldomain. In some embodiments, the effector protein may be a RNA-bindingprotein that cleaves a single strand of RNA. If the RNA bound is ssRNA,then the ssRNA is fully cleaved. In some embodiments, the effectorprotein may be a RNA-binding protein that cleaves a double strand ofRNA, for example if it comprises two RNase domains. If the RNA bound isdsRNA, then the dsRNA is fully cleaved.

RNase function in CRISPR systems is known, for example mRNA targetinghas been reported for certain type III CRISPR-Cas systems (Hale et al.,2014, Genes Dev, vol. 28, 2432-2443; Hale et al., 2009, Cell, vol. 139,945-956; Peng et al., 2015, Nucleic acids research, vol. 43, 406-417)and provides significant advantages. In the Staphylococcus epidermistype III-A system, transcription across targets results in cleavge ofthe target DNA and its transcripts, mediated by independent active siteswithin the Cas10-Csm ribonucleoprotein effector complex (see, Samai etal., 2015, Cell, vol. 151, 1164-1174). A CRISPR-Cas system, compositionor method targeting RNA via the present effector proteins is thusprovided.

The target RNA, i.e. the RNA of interest, is the RNA to be targeted bythe present invention leading to the recruitment to, and the binding ofthe effector protein at, the target site of interest on the target RNA.The target RNA may be any suitable form of RNA. This may include, insome embodiments, mRNA. In other embodiments, the target RNA may includetRNA or rRNA. In other embodiments, the target RNA may include miRNA. Inother embodiments, the target RNA may include siRNA.

Guide RNAs

As used herein, the term “guide sequence,” “crRNA” or “guide RNA” or“single guide RNA,” “gRNA” refers to a polynucleotide comprising anypolynucleotide sequence having sufficient complementarity with a targetnucleic acid sequence to hybridize with the target nucleic acid sequenceand to direct sequence-specific binding of a RNA-targeting complexcomprising the gRNA and a CRISPR effector protein to the target nucleicacid sequence. In general, a gRNA may be any polynucleotide sequence (i)being able to form a complex with a CRISPR effector protein and (ii)comprising a sequence having sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence. Asused herein the term “capable of forming a complex with the CRISPReffector protein” refers to the gRNA having a structure that allowsspecific binding by the CRISPR effector protein to the gRNA such that acomplex is formed that is capable of binding to a target RNA in asequence specific manner and that can exert a function on said targetRNA. Structural components of the gRNA may include direct repeats and aguide sequence (or spacer). The sequence specific binding to the targetRNA is mediated by a part of the gRNA, the “guide sequence”, beingcomplementary to the target RNA. In embodiments of the invention theterm guide RNA, i.e. RNA capable of guiding Cas to a target locus, isused as in foregoing cited documents such as WO 2014/093622(PCT/US2013/074667). As used herein the term “wherein the guide sequenceis capable of hybridizing” refers to a subsection of the gRNA havingsufficient complementarity to the target sequence to hybridize theretoand to mediate binding of a CRISPR complex to the target RNA. Ingeneral, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a CRISPR complex to the target sequence. In some embodiments, thedegree of complementarity between a guide sequence and its correspondingtarget sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT,Novoalign (Novocraft Technologies; available at novocraft.com), ELAND(Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn),and Maq (available at maq.sourceforge.net).

In certain embodiments, the CRISPR system as provided herein can makeuse of a crRNA or analogous polynucleotide comprising a guide sequence,wherein the polynucleotide is an RNA, a DNA or a mixture of RNA and DNA,and/or wherein the polynucleotide comprises one or more nucleotideanalogs. The sequence can comprise any structure, including but notlimited to a structure of a native crRNA, such as a bulge, a hairpin ora stem loop structure. In certain embodiments, the polynucleotidecomprising the guide sequence forms a duplex with a secondpolynucleotide sequence which can be an RNA or a DNA sequence.

In certain embodiments, use is made of chemically modified guide RNAs.Examples of guide RNA chemical modifications include, withoutlimitation, incorporation of 2′-O-methyl (M), 2′-O-methyl3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP) at one or moreterminal nucleotides. Such chemically modified guide RNAs can compriseincreased stability and increased activity as compared to unmodifiedguide RNAs, though on-target vs. off-target specificity is notpredictable. (See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi:10.1038/nbt.3290, published online 29 Jun. 2015). Chemically modifiedguide RNAs further include, without limitation, RNAs withphosphorothioate linkages and locked nucleic acid (LNA) nucleotidescomprising a methylene bridge between the 2′ and 4′ carbons of theribose ring.

In some embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10 to 30 nucleotides long. The ability of a guide sequenceto direct sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay. Similarly, cleavage of a target RNA may beevaluated in a test tube by providing the target sequence, components ofa CRISPR complex, including the guide sequence to be tested and acontrol guide sequence different from the test guide sequence, andcomparing binding or rate of cleavage at the target sequence between thetest and control guide sequence reactions. Other assays are possible,and will occur to those skilled in the art.

A guide sequence, and hence a nucleic acid-targeting guide RNA may beselected to target any target nucleic acid sequence. In the context offormation of a CRISPR complex, “target sequence” refers to a sequence towhich a guide sequence is designed to have complementarity, wherehybridization between a target sequence and a guide sequence promotesthe formation of a CRISPR complex. A target sequence may comprise RNApolynucleotides. The term “target RNA” refers to a RNA polynucleotidebeing or comprising the target sequence. In other words, the target RNAmay be a RNA polynucleotide or a part of a RNA polynucleotide to which apart of the gRNA, i.e. the guide sequence, is designed to havecomplementarity and to which the effector function mediated by thecomplex comprising CRISPR effector protein and a gRNA is to be directed.In some embodiments, a target sequence is located in the nucleus orcytoplasm of a cell. The target sequence may be DNA. The target sequencemay be any RNA sequence. In some embodiments, the target sequence may bea sequence within a RNA molecule selected from the group consisting ofmessenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA(tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclearRNA (snRNA), small nuclear RNA (snoRNA), double stranded RNA (dsRNA),non coding RNA (ncRNA), long non-coding RNA (lncRNA), and smallcytoplasmic RNA (scRNA). In some preferred embodiments, the targetsequence may be a sequence within a RNA molecule selected from the groupconsisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments,the target sequence may be a sequence within a RNA molecule selectedfrom the group consisting of ncRNA, and lncRNA. In some more preferredembodiments, the target sequence may be a sequence within an mRNAmolecule or a pre-mRNA molecule.

In some embodiments, a nucleic acid-targeting guide RNA is selected toreduce the degree of secondary structure within the RNA-targeting guideRNA. In some embodiments, about or less than about 75%, 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleicacid-targeting guide RNA participate in self-complementary base pairingwhen optimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carrand GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

In certain embodiments, a guide RNA or crRNA may comprise, consistessentially of, or consist of a direct repeat (DR) sequence and a guidesequence or spacer sequence. In certain embodiments, the guide RNA orcrRNA may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.In certain embodiments, the direct repeat sequence may be locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In otherembodiments, the direct repeat sequence may be located downstream (i.e.,3′) from the guide sequence or spacer sequence.

In certain embodiments, the crRNA comprises a stem loop, preferably asingle stem loop. In certain embodiments, the direct repeat sequenceforms a stem loop, preferably a single stem loop.

In certain embodiments, the spacer length (i.e. guide sequence lenth orspacer sequence length) of the guide RNA is from 15 to 35 nt. In certainembodiments, the spacer length of the guide RNA is from 18 to 35 nt. Incertain embodiments, the spacer length of the guide RNA is from 19 to 33nt. In certain embodiments, the spacer length of the guide RNA is from20 to 30 nt. In certain embodiments, the spacer length of the guide RNAis at least 15 nucleotides, preferably at least 18 nt, such as at least19, 20, 21, 22, or more nt. In certain embodiments, the spacer length isfrom 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17,18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26,or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt,e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.

In certain embidiments, the spacer length is 18 to 30 nucleotides. Incertain embodiments, the spacer length is 19 to 30 nucleotides. Incertain embodiments, the spacer length is 20 to 30 nucleotides. Incertain embodiments, the spacer length is 21 to 30 nucleotides. Incertain embodiments, the spacer length is 22 to 30 nucleotides. Incertain embodiments, the spacer length is 23 to 30 nucleotides. Incertain embodiments, the spacer length is 24 to 30 nucleotides. Incertain embodiments, the spacer length is 25 to 30 nucleotides. Incertain embodiments, the spacer length is 26 to 30 nucleotides. Incertain embodiments, the spacer length is 27 to 30 nucleotides. Incertain embodiments, the spacer length is 28 to 30 nucleotides. Incertain embodiments, the spacer length is 29 to 30 nucleotides. Incertain embidiments, the spacer length is 18 to 29 nucleotides. Incertain embodiments, the spacer length is 19 to 29 nucleotides. Incertain embodiments, the spacer length is 20 to 29 nucleotides. Incertain embodiments, the spacer length is 21 to 29 nucleotides. Incertain embodiments, the spacer length is 22 to 29 nucleotides. Incertain embodiments, the spacer length is 23 to 29 nucleotides. Incertain embodiments, the spacer length is 24 to 29 nucleotides. Incertain embodiments, the spacer length is 25 to 29 nucleotides. Incertain embodiments, the spacer length is 26 to 29 nucleotides. Incertain embodiments, the spacer length is 27 to 29 nucleotides. Incertain embodiments, the spacer length is 28 to 29 nucleotides. Incertain embidiments, the spacer length is 18 to 28 nucleotides. Incertain embodiments, the spacer length is 19 to 28 nucleotides. Incertain embodiments, the spacer length is 20 to 28 nucleotides. Incertain embodiments, the spacer length is 21 to 28 nucleotides. Incertain embodiments, the spacer length is 22 to 28 nucleotides. Incertain embodiments, the spacer length is 23 to 28 nucleotides. Incertain embodiments, the spacer length is 24 to 28 nucleotides. Incertain embodiments, the spacer length is 25 to 28 nucleotides. Incertain embodiments, the spacer length is 26 to 28 nucleotides. Incertain embodiments, the spacer length is 27 to 28 nucleotides. Incertain embidiments, the spacer length is 18 to 27 nucleotides. Incertain embodiments, the spacer length is 19 to 27 nucleotides. Incertain embodiments, the spacer length is 20 to 27 nucleotides. Incertain embodiments, the spacer length is 21 to 27 nucleotides. Incertain embodiments, the spacer length is 22 to 27 nucleotides. Incertain embodiments, the spacer length is 23 to 27 nucleotides. Incertain embodiments, the spacer length is 24 to 27 nucleotides. Incertain embodiments, the spacer length is 25 to 27 nucleotides. Incertain embodiments, the spacer length is 26 to 27 nucleotides. Incertain embidiments, the spacer length is 18 to 26 nucleotides. Incertain embodiments, the spacer length is 19 to 26 nucleotides. Incertain embodiments, the spacer length is 20 to 26 nucleotides. Incertain embodiments, the spacer length is 21 to 26 nucleotides. Incertain embodiments, the spacer length is 22 to 26 nucleotides. Incertain embodiments, the spacer length is 23 to 26 nucleotides. Incertain embodiments, the spacer length is 24 to 26 nucleotides. Incertain embodiments, the spacer length is 25 to 26 nucleotides. Incertain embidiments, the spacer length is 18 to 25 nucleotides. Incertain embodiments, the spacer length is 19 to 25 nucleotides. Incertain embodiments, the spacer length is 20 to 25 nucleotides. Incertain embodiments, the spacer length is 21 to 25 nucleotides. Incertain embodiments, the spacer length is 22 to 25 nucleotides. Incertain embodiments, the spacer length is 23 to 25 nucleotides. Incertain embodiments, the spacer length is 24 to 25 nucleotides. Incertain embidiments, the spacer length is 18 to 24 nucleotides. Incertain embodiments, the spacer length is 19 to 24 nucleotides. Incertain embodiments, the spacer length is 20 to 24 nucleotides. Incertain embodiments, the spacer length is 21 to 24 nucleotides. Incertain embodiments, the spacer length is 22 to 24 nucleotides. Incertain embodiments, the spacer length is 23 to 24 nucleotides. Incertain embidiments, the spacer length is 18 to 23 nucleotides. Incertain embodiments, the spacer length is 19 to 23 nucleotides. Incertain embodiments, the spacer length is 20 to 23 nucleotides. Incertain embodiments, the spacer length is 21 to 23 nucleotides. Incertain embodiments, the spacer length is 22 to 23 nucleotides. Incertain embidiments, the spacer length is 18 to 22 nucleotides. Incertain embodiments, the spacer length is 19 to 22 nucleotides. Incertain embodiments, the spacer length is 20 to 22 nucleotides. Incertain embodiments, the spacer length is 21 to 22 nucleotides. Incertain embidiments, the spacer length is 18 to 21 nucleotides. Incertain embodiments, the spacer length is 19 to 21 nucleotides. Incertain embodiments, the spacer length is 20 to 21 nucleotides. Incertain embidiments, the spacer length is 18 to 20 nucleotides. Incertain embodiments, the spacer length is 19 to 20 nucleotides. Incertain embidiments, the spacer length is 18 to 19 nucleotides.

In certain embodiments, the spacer length of the guide RNA is less than28 nucleotides. In certain embodiments, the spacer length of the guideRNA is at least 18 nucleotides and less than 28 nucleotides. In certainembodiments, the spacer length of the guide RNA is between 19 and 28nucleotides. In certain embodiments, the spacer length of the guide RNAis between 19 and 25 nucleotides. In certain embodiments, the spacerlength of the guide RNA is 20 nucleotides. In certain embodiments, thespacer length of the guide RNA is 23 nucleotides. In certainembodiments, the spacer length of the guide RNA is 25 nucleotides.

In a classic CRISPR-Cas systems, the degree of complementarity between aguide sequence and its corresponding target sequence can be about ormore than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%;a guide or RNA or sgRNA can be about or more than about 5, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA orsgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, orfewer nucleotides in length. However, an aspect of the invention is toreduce off-target interactions, e.g., reduce the guide interacting witha target sequence having low complementarity. Indeed, in the examples,it is shown that the invention involves mutations that result in theCRISPR-Cas system being able to distinguish between target andoff-target sequences that have greater than 80% to about 95%complementarity, e.g., 83%-84% or 88-89% or 94-95% complementarity (forinstance, distinguishing between a target having 18 nucleotides from anoff-target of 18 nucleotides having 1, 2 or 3 mismatches). Accordingly,in the context of the present invention the degree of complementaritybetween a guide sequence and its corresponding target sequence isgreater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or98% or 98.5% or 99% or 99.5% or 99.9%, or 100%. Off target is less than100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90%or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80%complementarity between the sequence and the guide, with it advantageousthat off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98%or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementaritybetween the sequence and the guide.

In certain embodiments, modulations of cleavage efficiency can beexploited by introduction of mismatches, e.g. 1 or more mismatches, suchas 1 or 2 mismatches between spacer sequence and target sequence,including the position of the mismatch along the spacer/target. The morecentral (i.e. not 3′ or 5′) for instance a double mismatch is, the morecleavage efficiency is affected. Accordingly, by choosing mismatchposition along the spacer, cleavage efficiency can be modulated. Bymeans of example, if less than 100% cleavage of targets is desired (e.g.in a cell population), 1 or more, such as preferably 2 mismatchesbetween spacer and target sequence may be introduced in the spacersequences. The more central along the spacer of the mismatch position,the lower the cleavage percentage.

In certain example embodiments, the cleavage efficiency may be exploitedto design single guides that can distinguish two or more targets thatvary by a single nucleotide, such as a single nucleotide polymorphism(SNP), variation, or (point) mutation. This aspect is of particularrelevance for the diagnostic applications of the present invention, suchas in particular the companion diagnostic applications. The CRISPReffector may have reduced sensitivity to SNPs (or other singlenucleotide variations) and continue to cleave SNP targets with a certainlevel of efficiency. Thus, for two targets, or a set of targets, a guideRNA may be designed with a nucleotide sequence that is complementary toone of the targets i.e. the on-target SNP. The guide RNA is furtherdesigned to have a synthetic mismatch. As used herein a “syntheticmismatch” refers to a non-naturally occurring mismatch that isintroduced upstream or downstream of the naturally occurring SNP, suchas at most 5 nucleotides upstream or downstream, for instance 4, 3, 2,or 1 nucleotide upstream or downstream, preferably at most 3 nucleotidesupstream or downstream, more preferably at most 2 nucleotides upstreamor downstream, most preferably 1 nucleotide upstream or downstream (i.e.adjacent the SNP). When the CRISPR effector binds to the on-target SNP,only a single mismatch will be formed with the synthetic mismatch andthe CRISPR effector will continue to be activated and a detectablesignal produced. When the guide RNA hybridizes to an off-target SNP, twomismatches will be formed, the mismatch from the SNP and the syntheticmismatch, and no detectable signal generated. Thus, the systemsdisclosed herein may be designed to distinguish SNPs within apopulation. For, example the systems may be used to distinguishpathogenic strains that differ by a single SNP or detect certain diseasespecific SNPs, such as but not limited to, disease associated SNPs, suchas without limitation cancer associated SNPs.

In certain embodiments, the guide RNA is designed such that the SNP islocated on position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of thespacer sequence (starting at the 5′ end). In certain embodiments, theguide RNA is designed such that the SNP is located on position 1, 2, 3,4, 5, 6, 7, 8, or 9 of the spacer sequence (starting at the 5′ end). Incertain embodiments, the guide RNA is designed such that the SNP islocated on position 2, 3, 4, 5, 6, or 7 of the spacer sequence (startingat the 5′ end). In certain embodiments, the guide RNA is designed suchthat the SNP is located on position 3, 4, 5, or 6 of the spacer sequence(starting at the 5′ end). In certain embodiments, the guide RNA isdesigned such that the SNP is located on position 3 of the spacersequence (starting at the 5′ end).

In certain embodiments, the guide RNA is designed such that the mismatch(e.g. the synthetic mismatch, i.e. an additional mutation besides a SNP)is located on position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of thespacer sequence (starting at the 5′ end). In certain embodiments, theguide RNA is designed such that the mismatch is located on position 1,2, 3, 4, 5, 6, 7, 8, or 9 of the spacer sequence (starting at the 5′end). In certain embodiments, the guide RNA is designed such that themismatch is located on position 4, 5, 6, or 7 of the spacer sequence(starting at the 5′ end. In certain embodiments, the guide RNA isdesigned such that the mismatch is located on position 5 of the spacersequence (starting at the 5′ end).

In certain embodiments, the guide RNA is designed such that the mismatchis located 2 nucleotides upstream of the SNP (i.e. one interveningnucleotide).

In certain embodiments, the guide RNA is designed such that the mismatchis located 2 nucleotides downstream of the SNP (i.e. one interveningnucleotide).

In certain embodiments, the guide RNA is designed such that the mismatchis located on position 5 of the spacer sequence (starting at the 5′ end)and the SNP is located on position 3 of the spacer sequence (starting atthe 5′ end).

The embodiments described herein comprehend inducing one or morenucleotide modifications in a eukaryotic cell (in vitro, i.e. in anisolated eukaryotic cell) as herein discussed comprising delivering tocell a vector as herein discussed. The mutation(s) can include theintroduction, deletion, or substitution of one or more nucleotides ateach target sequence of cell(s) via the guide(s) RNA(s). The mutationscan include the introduction, deletion, or substitution of 1-75nucleotides at each target sequence of said cell(s) via the guide(s)RNA(s). The mutations can include the introduction, deletion, orsubstitution of 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides ateach target sequence of said cell(s) via the guide(s) RNA(s). Themutations can include the introduction, deletion, or substitution of 5,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence ofsaid cell(s) via the guide(s) RNA(s). The mutations include theintroduction, deletion, or substitution of 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,or 75 nucleotides at each target sequence of said cell(s) via theguide(s) RNA(s). The mutations can include the introduction, deletion,or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, or 75 nucleotides at each target sequence of said cell(s) viathe guide(s) RNA(s). The mutations can include the introduction,deletion, or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500nucleotides at each target sequence of said cell(s) via the guide(s)RNA(s).

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50,or more base pairs from) the target sequence, but may depend on forinstance secondary structure, in particular in the case of RNA targets.

Guide RNA According to the Invention Comprising a Dead Guide Sequence

In one aspect, the invention provides guide sequences which are modifiedin a manner which allows for formation of the CRISPR complex andsuccessful binding to the target, while at the same time, not allowingfor successful nuclease activity (i.e. without nuclease activity/withoutindel activity). For matters of explanation such modified guidesequences are referred to as “dead guides” or “dead guide sequences”.These dead guides or dead guide sequences can be thought of ascatalytically inactive or conformationally inactive with regard tonuclease activity. Indeed, dead guide sequences may not sufficientlyengage in productive base pairing with respect to the ability to promotecatalytic activity or to distinguish on-target and off-target bindingactivity. Briefly, the assay involves synthesizing a CRISPR target RNAand guide RNAs comprising mismatches with the target RNA, combiningthese with the RNA targeting enzyme and analyzing cleavage based on gelsbased on the presence of bands generated by cleavage products, andquantifying cleavage based upon relative band intensities.

Hence, in a related aspect, the invention provides a non-naturallyoccurring or engineered composition RNA targeting CRISPR-Cas systemcomprising a functional RNA targeting as described herein, and guide RNA(gRNA) wherein the gRNA comprises a dead guide sequence whereby the gRNAis capable of hybridizing to a target sequence such that the RNAtargeting CRISPR-Cas system is directed to a genomic locus of interestin a cell without detectable RNA cleavage activity of a non-mutant RNAtargeting enzyme of the system. It is to be understood that any of thegRNAs according to the invention as described herein elsewhere may beused as dead gRNAs/gRNAs comprising a dead guide sequence as describedherein below. Any of the methods, products, compositions and uses asdescribed herein elsewhere is equally applicable with the deadgRNAs/gRNAs comprising a dead guide sequence as further detailed below.By means of further guidance, the following particular aspects andembodiments are provided.

The ability of a dead guide sequence to direct sequence-specific bindingof a CRISPR complex to an RNA target sequence may be assessed by anysuitable assay. For example, the components of a CRISPR systemsufficient to form a CRISPR complex, including the dead guide sequenceto be tested, may be provided to a host cell having the correspondingtarget sequence, such as by transfection with vectors encoding thecomponents of the CRISPR sequence, followed by an assessment ofpreferential cleavage within the target sequence. For instance, cleavageof a target RNA polynucleotide sequence may be evaluated in a test tubeby providing the target sequence, components of a CRISPR complex,including the dead guide sequence to be tested and a control guidesequence different from the test dead guide sequence, and comparingbinding or rate of cleavage at the target sequence between the test andcontrol guide sequence reactions. Other assays are possible, and willoccur to those skilled in the art. A dead guide sequence may be selectedto target any target sequence. In some embodiments, the target sequenceis a sequence within a genome of a cell.

As explained further herein, several structural parameters allow for aproper framework to arrive at such dead guides. Dead guide sequences aretypically shorter than respective guide sequences which result in activeRNA cleavage. In particular embodiments, dead guides are 5%, 10%, 20%,30%, 40%, 50%, shorter than respective guides directed to the same.

As explained below and known in the art, one aspect of gRNA-RNAtargeting specificity is the direct repeat sequence, which is to beappropriately linked to such guides. In particular, this implies thatthe direct repeat sequences are designed dependent on the origin of theRNA targeting enzyme. Thus, structural data available for validated deadguide sequences may be used for designing CRISPR protein specificequivalents. Structural similarity between, e.g., the orthologousnuclease domains HEPN of two or more CRISPR effector proteins may beused to transfer design equivalent dead guides. Thus, the dead guideherein may be appropriately modified in length and sequence to reflectsuch CRISPR protein specific equivalents, allowing for formation of theCRISPR complex and successful binding to the target RNA, while at thesame time, not allowing for successful nuclease activity.

The use of dead guides in the context herein as well as the state of theart provides a surprising and unexpected platform for network biologyand/or systems biology in both in vitro, ex vivo, and in vivoapplications, allowing for multiplex gene targeting, and in particularbidirectional multiplex gene targeting. Prior to the use of dead guides,addressing multiple targets has been challenging and in some cases notpossible. With the use of dead guides, multiple targets, and thusmultiple activities, may be addressed, for example, in the same cell, inthe same animal, or in the same patient. Such multiplexing may occur atthe same time or staggered for a desired timeframe.

For example, the dead guides allow to use gRNA as a means for genetargeting, without the consequence of nuclease activity, while at thesame time providing directed means for activation or repression. GuideRNA comprising a dead guide may be modified to further include elementsin a manner which allow for activation or repression of gene activity,in particular protein adaptors (e.g. aptamers) as described hereinelsewhere allowing for functional placement of gene effectors (e.g.activators or repressors of gene activity). One example is theincorporation of aptamers, as explained herein and in the state of theart. By engineering the gRNA comprising a dead guide to incorporateprotein-interacting aptamers (Konermann et al., “Genome-scaletranscription activation by an engineered CRISPR-Cas9 complex,”doi:10.1038/nature14136, incorporated herein by reference), one mayassemble multiple distinct effector domains. Such may be modeled afternatural processes.

Thus, one aspect is a gRNA of the invention which comprises a deadguide, wherein the gRNA further comprises modifications which providefor gene activation or repression, as described herein. The dead gRNAmay comprise one or more aptamers. The aptamers may be specific to geneeffectors, gene activators or gene repressors. Alternatively, theaptamers may be specific to a protein which in turn is specific to andrecruits/binds a specific gene effector, gene activator or generepressor. If there are multiple sites for activator or repressorrecruitment, it is preferred that the sites are specific to eitheractivators or repressors. If there are multiple sites for activator orrepressor binding, the sites may be specific to the same activators orsame repressors. The sites may also be specific to different activatorsor different repressors. The effectors, activators, repressors may bepresent in the form of fusion proteins.

In an aspect, the invention provides a method of selecting a dead guideRNA targeting sequence for directing a functionalized CRISPR system to agene locus in an organism, which comprises: a) locating one or moreCRISPR motifs in the gene locus; b) analyzing the 20 nt sequencedownstream of each CRISPR motif by: i) determining the GC content of thesequence; and ii) determining whether there are off-target matches ofthe first 15 nt of the sequence in the genome of the organism; c)selecting the sequence for use in a guide RNA if the GC content of thesequence is 70% or less and no off-target matches are identified. In anembodiment, the sequence is selected if the GC content is 50% or less.In an embodiment, the sequence is selected if the GC content is 40% orless. In an embodiment, the sequence is selected if the GC content is30% or less. In an embodiment, two or more sequences are analyzed andthe sequence having the lowest GC content is selected. In an embodiment,off-target matches are determined in regulatory sequences of theorganism. In an embodiment, the gene locus is a regulatory region. Anaspect provides a dead guide RNA comprising the targeting sequenceselected according to the aforementioned methods.

In an aspect, the invention provides a dead guide RNA for targeting afunctionalized CRISPR system to a gene locus in an organism. In anembodiment of the invention, the dead guide RNA comprises a targetingsequence wherein the CG content of the target sequence is 70% or less,and the first 15 nt of the targeting sequence does not match anoff-target sequence downstream from a CRISPR motif in the regulatorysequence of another gene locus in the organism. In certain embodiments,the GC content of the targeting sequence 60% or less, 55% or less, 50%or less, 45% or less, 40% or less, 35% or less or 30% or less. Incertain embodiments, the GC content of the targeting sequence is from70% to 60% or from 60% to 50% or from 50% to 40% or from 40% to 30%. Inan embodiment, the targeting sequence has the lowest CG content amongpotential targeting sequences of the locus.

In an embodiment of the invention, the first 15 nt of the dead guidematch the target sequence. In another embodiment, first 14 nt of thedead guide match the target sequence. In another embodiment, the first13 nt of the dead guide match the target sequence. In another embodimentfirst 12 nt of the dead guide match the target sequence. In anotherembodiment, first 11 nt of the dead guide match the target sequence. Inanother embodiment, the first 10 nt of the dead guide match the targetsequence. In an embodiment of the invention the first 15 nt of the deadguide does not match an off-target sequence downstream from a CRISPRmotif in the regulatory region of another gene locus. In otherembodiments, the first 14 nt, or the first 13 nt of the dead guide, orthe first 12 nt of the guide, or the first 11 nt of the dead guide, orthe first 10 nt of the dead guide, does not match an off-target sequencedownstream from a CRISPR motif in the regulatory region of another genelocus. In other embodiments, the first 15 nt, or 14 nt, or 13 nt, or 12nt, or 11 nt of the dead guide do not match an off-target sequencedownstream from a CRISPR motif in the genome.

In certain embodiments, the dead guide RNA includes additionalnucleotides at the 3′-end that do not match the target sequence. Thus, adead guide RNA that includes the first 20-28 nt, downstream of a CRISPRmotif can be extended in length at the 3′ end.

Vectors and Expression Systems

In certain aspects the invention involves vectors, e.g. for deliveringor introducing in a cell CRISPR effector and/or RNA capable of guidingCRISPR effector to a target locus (i.e. guide RNA), and optionally alsofor propagating these components (e.g. in prokaryotic cells). A usedherein, a “vector” is a tool that allows or facilitates the transfer ofan entity from one environment to another. It is a replicon, such as aplasmid, phage, or cosmid, into which another DNA segment may beinserted so as to bring about the replication of the inserted segment.Generally, a vector is capable of replication when associated with theproper control elements. In general, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g. circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses(AAVs)). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g. bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety. Thus, the embodiments disclosed herein mayalso comprise transgenic cells comprising the CRISPR effector system. Incertain example embodiments, the transgenic cell may function as anindividual discrete volume. In other words samples comprising a maskingconstruct may be delivered to a cell, for example in a suitable deliveryvesicle and if the target is present in the delivery vesicle the CRISPReffector is activated and a detectable signal generated.

The vector(s) can include the regulatory element(s), e.g., promoter(s).The vector(s) can comprise CRISPR effector encoding sequences, and/or asingle, but possibly also can comprise at least 2, 3, 4, 5, 6, 7, or 8or more, such as 10, 12, 14, 16 or more, such as 32 or 48 or 50 guideRNA(s) (e.g., sgRNAs) encoding sequences, such as 1-2, 1-3, 1-4 1-5,3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s)(e.g., sgRNAs). In a single vector there can be a promoter for each RNA(e.g., sgRNA), advantageously when there are up to about 16 RNA(s); and,when a single vector provides for more than 16 RNA(s), one or morepromoter(s) can drive expression of more than one of the RNA(s), e.g.,when there are 32 RNA(s), each promoter can drive expression of twoRNA(s), and when there are 48 RNA(s), each promoter can drive expressionof three RNA(s). By simple arithmetic and well established cloningprotocols and the teachings in this disclosure one skilled in the artcan readily practice the invention as to the RNA(s) for a suitableexemplary vector such as AAV, and a suitable promoter such as the U6promoter. For example, the packaging limit of AAV is ˜4.7 kb. The lengthof a single U6-gRNA (plus restriction sites for cloning) is 361 bp.Therefore, the skilled person can readily fit about 12-16, e.g., 13U6-gRNA cassettes in a single vector. This can be assembled by anysuitable means, such as a golden gate strategy used for TALE assembly(genome-engineering.org/taleffectors/). The skilled person can also usea tandem guide strategy to increase the number of U6-gRNAs byapproximately 1.5 times, e.g., to increase from 12-16, e.g., 13 toapproximately 18-24, e.g., about 19 U6-gRNAs. Therefore, one skilled inthe art can readily reach approximately 18-24, e.g., about 19promoter-RNAs, e.g., U6-gRNAs in a single vector, e.g., an AAV vector. Afurther means for increasing the number of promoters and RNAs in avector is to use a single promoter (e.g., U6) to express an array ofRNAs separated by cleavable sequences. And an even further means forincreasing the number of promoter-RNAs in a vector, is to express anarray of promoter-RNAs separated by cleavable sequences in the intron ofa coding sequence or gene; and, in this instance it is advantageous touse a polymerase II promoter, which can have increased expression andenable the transcription of long RNA in a tissue specific manner. (see,e.g., nar.oxfordjournals.org/content/34/7/e53.short andnature.com/mt/journal/v16/n9/abs/mt2008144a.html). In an advantageousembodiment, AAV may package U6 tandem gRNA targeting up to about 50genes. Accordingly, from the knowledge in the art and the teachings inthis disclosure the skilled person can readily make and use vector(s),e.g., a single vector, expressing multiple RNAs or guides under thecontrol or operatively or functionally linked to one or morepromoters-especially as to the numbers of RNAs or guides discussedherein, without any undue experimentation.

The guide RNA(s) encoding sequences and/or CRISPR effector encodingsequences, can be functionally or operatively linked to regulatoryelement(s) and hence the regulatory element(s) drive expression. Thepromoter(s) can be constitutive promoter(s) and/or conditionalpromoter(s) and/or inducible promoter(s) and/or tissue specificpromoter(s). The promoter can be selected from the group consisting ofRNA polymerases, pol I, pol II, pol III, T7, U6, H1, retroviral Roussarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter,the SV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter. An advantageous promoter is the promoter is U6.

In some embodiments, one or more elements of a nucleic acid-targetingsystem is derived from a particular organism comprising an endogenousCRISPR RNA-targeting system. In certain example embodiments, theeffector protein CRISPR RNA-targeting system comprises at least one HEPNdomain, including but not limited to the HEPN domains described herein,HEPN domains known in the art, and domains recognized to be HEPN domainsby comparison to consensus sequence motifs. Several such domains areprovided herein. In one non-limiting example, a consensus sequence canbe derived from the sequences of C2c2 or Cas13b orthologs providedherein. In certain example embodiments, the effector protein comprises asingle HEPN domain. In certain other example embodiments, the effectorprotein comprises two HEPN domains.

The term “nucleic acid-targeting system”, wherein nucleic acid is DNA orRNA, and in some aspects may also refer to DNA-RNA hybrids orderivatives thereof, refers collectively to transcripts and otherelements involved in the expression of or directing the activity of DNAor RNA-targeting CRISPR-associated (“Cas”) genes, which may includesequences encoding a DNA or RNA-targeting Cas protein and a DNA orRNA-targeting guide RNA comprising a CRISPR RNA (crRNA) sequence and (insome but not all systems) a trans-activating CRISPR/Cas system RNA(tracrRNA) sequence, or other sequences and transcripts from a DNA orRNA-targeting CRISPR locus. In general, a RNA-targeting system ischaracterized by elements that promote the formation of a DNA orRNA-targeting complex at the site of a target DNA or RNA sequence. Inthe context of formation of a DNA or RNA-targeting complex, “targetsequence” refers to a DNA or RNA sequence to which a DNA orRNA-targeting guide RNA is designed to have complementarity, wherehybridization between a target sequence and a RNA-targeting guide RNApromotes the formation of a RNA-targeting complex. In some embodiments,a target sequence is located in the nucleus or cytoplasm of a cell.

In an aspect of the invention, novel RNA targeting systems also referredto as RNA- or RNA-targeting CRISPR/Cas or the CRISPR-Cas systemRNA-targeting system of the present application are based on identifiedType VI Cas proteins which do not require the generation of customizedproteins to target specific RNA sequences but rather a single enzyme canbe programmed by a RNA molecule to recognize a specific RNA target, inother words the enzyme can be recruited to a specific RNA target usingsaid RNA molecule.

In an aspect of the invention, novel DNA targeting systems also referredto as DNA- or DNA-targeting CRISPR/Cas or the CRISPR-Cas systemRNA-targeting system of the present application are based on identifiedType VI Cas proteins which do not require the generation of customizedproteins to target specific RNA sequences but rather a single enzyme canbe programmed by a RNA molecule to recognize a specific DNA target, inother words the enzyme can be recruited to a specific DNA target usingsaid RNA molecule.

The nucleic acids-targeting systems, the vector systems, the vectors andthe compositions described herein may be used in various nucleicacids-targeting applications, altering or modifying synthesis of a geneproduct, such as a protein, nucleic acids cleavage, nucleic acidsediting, nucleic acids splicing; trafficking of target nucleic acids,tracing of target nucleic acids, isolation of target nucleic acids,visualization of target nucleic acids, etc.

In some embodiments, one or more vectors driving expression of one ormore elements of a nucleic acid-targeting system are introduced into ahost cell such that expression of the elements of the nucleicacid-targeting system direct formation of a nucleic acid-targetingcomplex at one or more target sites. For example, a nucleicacid-targeting effector enzyme and a nucleic acid-targeting guide RNAcould each be operably linked to separate regulatory elements onseparate vectors. RNA(s) of the nucleic acid-targeting system can bedelivered to a transgenic nucleic acid-targeting effector protein animalor mammal, e.g., an animal or mammal that constitutively or inducibly orconditionally expresses nucleic acid-targeting effector protein; or ananimal or mammal that is otherwise expressing nucleic acid-targetingeffector protein or has cells containing nucleic acid-targeting effectorprotein, such as by way of prior administration thereto of a vector orvectors that code for and express in vivo nucleic acid-targetingeffector protein. Alternatively, two or more of the elements expressedfrom the same or different regulatory elements, may be combined in asingle vector, with one or more additional vectors providing anycomponents of the nucleic acid-targeting system not included in thefirst vector. nucleic acid-targeting system elements that are combinedin a single vector may be arranged in any suitable orientation, such asone element located 5′ with respect to (“upstream” of) or 3′ withrespect to (“downstream” of) a second element. The coding sequence ofone element may be located on the same or opposite strand of the codingsequence of a second element, and oriented in the same or oppositedirection. In some embodiments, a single promoter drives expression of atranscript encoding a nucleic acid-targeting effector protein and thenucleic acid-targeting guide RNA, embedded within one or more intronsequences (e.g., each in a different intron, two or more in at least oneintron, or all in a single intron). In some embodiments, the nucleicacid-targeting effector protein and the nucleic acid-targeting guide RNAmay be operably linked to and expressed from the same promoter. Deliveryvehicles, vectors, particles, nanoparticles, formulations and componentsthereof for expression of one or more elements of a nucleicacid-targeting system are as used in the foregoing documents, such as WO2014/093622 (PCT/US2013/074667). In some embodiments, a vector comprisesone or more insertion sites, such as a restriction endonucleaserecognition sequence (also referred to as a “cloning site”). In someembodiments, one or more insertion sites (e.g., about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are locatedupstream and/or downstream of one or more sequence elements of one ormore vectors. In some embodiments, a vector comprises two or moreinsertion sites, so as to allow insertion of a guide sequence at eachsite. In such an arrangement, the two or more guide sequences maycomprise two or more copies of a single guide sequence, two or moredifferent guide sequences, or combinations of these. When multipledifferent guide sequences are used, a single expression construct may beused to target nucleic acid-targeting activity to multiple different,corresponding target sequences within a cell. For example, a singlevector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or more guide sequences. In some embodiments, about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more suchguide-sequence-containing vectors may be provided, and optionallydelivered to a cell. In some embodiments, a vector comprises aregulatory element operably linked to an enzyme-coding sequence encodinga a nucleic acid-targeting effector protein. nucleic acid-targetingeffector protein or nucleic acid-targeting guide RNA or RNA(s) can bedelivered separately; and advantageously at least one of these isdelivered via a particle or nanoparticle complex. nucleic acid-targetingeffector protein mRNA can be delivered prior to the nucleicacid-targeting guide RNA to give time for nucleic acid-targetingeffector protein to be expressed. nucleic acid-targeting effectorprotein mRNA might be administered 1-12 hours (preferably around 2-6hours) prior to the administration of nucleic acid-targeting guide RNA.Alternatively, nucleic acid-targeting effector protein mRNA and nucleicacid-targeting guide RNA can be administered together. Advantageously, asecond booster dose of guide RNA can be administered 1-12 hours(preferably around 2-6 hours) after the initial administration ofnucleic acid-targeting effector protein mRNA+guide RNA. Additionaladministrations of nucleic acid-targeting effector protein mRNA and/orguide RNA might be useful to achieve the most efficient levels of genomeand/or transcriptome modification.

In one aspect, the invention provides methods for using one or moreelements of a nucleic acid-targeting system. The nucleic acid-targetingcomplex of the invention provides an effective means for modifying atarget RNA. The nucleic acid-targeting complex of the invention has awide variety of utility including modifying (e.g., deleting, inserting,translocating, inactivating, activating) a target RNA in a multiplicityof cell types. As such the nucleic acid-targeting complex of theinvention has a broad spectrum of applications in, e.g., gene therapy,drug screening, disease diagnosis, and prognosis. An exemplary nucleicacid-targeting complex comprises a RNA-targeting effector proteincomplexed with a guide RNA hybridized to a target sequence within thetarget locus of interest.

In one embodiment, this invention provides a method of cleaving a targetRNA. The method may comprise modifying a target RNA using a nucleicacid-targeting complex that binds to the target RNA and effect cleavageof said target RNA. In an embodiment, the nucleic acid-targeting complexof the invention, when introduced into a cell, may create a break (e.g.,a single or a double strand break) in the RNA sequence. For example, themethod can be used to cleave a disease RNA in a cell. For example, anexogenous RNA template comprising a sequence to be integrated flanked byan upstream sequence and a downstream sequence may be introduced into acell. The upstream and downstream sequences share sequence similaritywith either side of the site of integration in the RNA. Where desired, adonor RNA can be mRNA. The exogenous RNA template comprises a sequenceto be integrated (e.g., a mutated RNA). The sequence for integration maybe a sequence endogenous or exogenous to the cell. Examples of asequence to be integrated include RNA encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction. The upstream and downstream sequences in the exogenous RNAtemplate are selected to promote recombination between the RNA sequenceof interest and the donor RNA. The upstream sequence is a RNA sequencethat shares sequence similarity with the RNA sequence upstream of thetargeted site for integration. Similarly, the downstream sequence is aRNA sequence that shares sequence similarity with the RNA sequencedownstream of the targeted site of integration. The upstream anddownstream sequences in the exogenous RNA template can have 75%, 80%,85%, 90%, 95%, or 100% sequence identity with the targeted RNA sequence.Preferably, the upstream and downstream sequences in the exogenous RNAtemplate have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identitywith the targeted RNA sequence. In some methods, the upstream anddownstream sequences in the exogenous RNA template have about 99% or100% sequence identity with the targeted RNA sequence. An upstream ordownstream sequence may comprise from about 20 bp to about 2500 bp, forexample, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, or 2500 bp. In some methods, the exemplary upstream ordownstream sequence have about 200 bp to about 2000 bp, about 600 bp toabout 1000 bp, or more particularly about 700 bp to about 1000 bp. Insome methods, the exogenous RNA template may further comprise a marker.Such a marker may make it easy to screen for targeted integrations.Examples of suitable markers include restriction sites, fluorescentproteins, or selectable markers. The exogenous RNA template of theinvention can be constructed using recombinant techniques (see, forexample, Sambrook et al., 2001 and Ausubel et al., 1996). In a methodfor modifying a target RNA by integrating an exogenous RNA template, abreak (e.g., double or single stranded break in double or singlestranded DNA or RNA) is introduced into the DNA or RNA sequence by thenucleic acid-targeting complex, the break is repaired via homologousrecombination with an exogenous RNA template such that the template isintegrated into the RNA target. The presence of a double-stranded breakfacilitates integration of the template. In other embodiments, thisinvention provides a method of modifying expression of a RNA in aeukaryotic cell. The method comprises increasing or decreasingexpression of a target polynucleotide by using a nucleic acid-targetingcomplex that binds to the RNA (e.g., mRNA or pre-mRNA). In some methods,a target RNA can be inactivated to effect the modification of theexpression in a cell. For example, upon the binding of a RNA-targetingcomplex to a target sequence in a cell, the target RNA is inactivatedsuch that the sequence is not translated, the coded protein is notproduced, or the sequence does not function as the wild-type sequencedoes. For example, a protein or microRNA coding sequence may beinactivated such that the protein or microRNA or pre-microRNA transcriptis not produced. The target RNA of a RNA-targeting complex can be anyRNA endogenous or exogenous to the eukaryotic cell. For example, thetarget RNA can be a RNA residing in the nucleus of the eukaryotic cell.The target RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA). Examples of target RNA include a sequence associatedwith a signaling biochemical pathway, e.g., a signaling biochemicalpathway-associated RNA. Examples of target RNA include a diseaseassociated RNA. A “disease-associated” RNA refers to any RNA which isyielding translation products at an abnormal level or in an abnormalform in cells derived from a disease-affected tissues compared withtissues or cells of a non disease control. It may be a RNA transcribedfrom a gene that becomes expressed at an abnormally high level; it maybe a RNA transcribed from a gene that becomes expressed at an abnormallylow level, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated RNA also refersto a RNA transcribed from a gene possessing mutation(s) or geneticvariation that is directly responsible or is in linkage disequilibriumwith a gene(s) that is responsible for the etiology of a disease. Thetranslated products may be known or unknown, and may be at a normal orabnormal level. The target RNA of a RNA-targeting complex can be any RNAendogenous or exogenous to the eukaryotic cell. For example, the targetRNA can be a RNA residing in the nucleus of the eukaryotic cell. Thetarget RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, IncRNA,tRNA, or rRNA).

In some embodiments, the method may comprise allowing a nucleicacid-targeting complex to bind to the target RNA to effect cleavage ofsaid target RNA or RNA thereby modifying the target RNA, wherein thenucleic acid-targeting complex comprises a nucleic acid-targetingeffector protein complexed with a guide RNA hybridized to a targetsequence within said target RNA. In one aspect, the invention provides amethod of modifying expression of RNA in a eukaryotic cell. In someembodiments, the method comprises allowing a nucleic acid-targetingcomplex to bind to the RNA such that said binding results in increasedor decreased expression of said RNA; wherein the nucleic acid-targetingcomplex comprises a nucleic acid-targeting effector protein complexedwith a guide RNA. Similar considerations and conditions apply as abovefor methods of modifying a target RNA. In fact, these sampling,culturing and re-introduction options apply across the aspects of thepresent invention. In one aspect, the invention provides for methods ofmodifying a target RNA in a eukaryotic cell, which may be in vivo, exvivo or in vitro. In some embodiments, the method comprises sampling acell or population of cells from a human or non-human animal, andmodifying the cell or cells. Culturing may occur at any stage ex vivo.The cell or cells may even be re-introduced into the non-human animal orplant. For re-introduced cells it is particularly preferred that thecells are stem cells.

Indeed, in any aspect of the invention, the nucleic acid-targetingcomplex may comprise a nucleic acid-targeting effector protein complexedwith a guide RNA hybridized to a target sequence.

The invention relates to the engineering and optimization of systems,methods and compositions used for the control of gene expressioninvolving RNA sequence targeting, that relate to the nucleicacid-targeting system and components thereof. In advantageousembodiments, the effector protein enzyme is a Type VI protein such asC2c2. An advantage of the present methods is that the CRISPR systemminimizes or avoids off-target binding and its resulting side effects.This is achieved using systems arranged to have a high degree ofsequence specificity for the target RNA.

In other example embodiments, the Type VI RNA-targeting Cas enzyme isCas 13d. In certain embodiments, Cas13d is Eubacterium siraeum DSM 15702(EsCas13d) or Ruminococcus sp. N15. MGS-57 (RspCas13d) (see, e.g., Yanet al., Cas13d Is a Compact RNA-Targeting Type VI CRISPR EffectorPositively Modulated by a WYL-Domain-Containing Accessory Protein,Molecular Cell (2018), doi.org/10.1016/j.molcel.2018.02.028). RspCas13dand EsCas13d have no flanking sequence requirements (e.g., PFS, PAM).

Delivery Generally CRISPR Effector Protein Complexes can DeliverFunctional Effectors

Unlike CRISPR-Cas-mediated gene knockout, which permanently eliminatesexpression by mutating the gene at the DNA level, CRISPR-Cas knockdownallows for temporary reduction of gene expression through the use ofartificial transcription or translation factors. Mutating key residuesin both DNA or RNA cleavage domains of the CRISPR protein results in thegeneration of a catalytically inactive CRISPR protein. A catalyticallyinactive CRISPR complexes with a guide RNA and localizes to the or RNAsequence specified by that guide RNA's targeting domain, however, itdoes not cleave the target RNA. Fusion of the inactive CRISPR protein toan effector domain, e.g., a transcription or translation repressiondomain, enables recruitment of the effector to any or RNA site specifiedby the guide RNA. In certain embodiments, CRISPR effector may be fusedto a transcriptional repression domain and recruited to the promoterregion of a gene. Especially for gene repression, it is contemplatedherein that blocking the binding site of an endogenous transcriptionfactor would aid in downregulating gene expression. In anotherembodiment, an inactive CRISPR protein can be fused to a chromatinmodifying protein. Altering chromatin status can result in decreasedexpression of the target gene. In further embodiments, CRISPR proteinmay be fused to a translation repression domain.

In an embodiment, a guide RNA molecule can be targeted to a knowntranscription response elements (e.g., promoters, enhancers, etc.), aknown upstream activating sequences, and/or sequences of unknown orknown function that are suspected of being able to control (protein)expression of the target RNA.

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does. For example, a protein or microRNA codingsequence may be inactivated such that the protein is not produced. Insome methods, a target polynucleotide can be inactivated to effect themodification of the expression in a cell. For example, upon the bindingof a CRISPR complex to an RNA target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not translated,affecting the expression level of the protein in the cell.

In particular embodiments, the CRISPR enzyme comprises one or moremutations selected from the group consisting of R597A, H602A, R1278A andH1283A and/or the one or more mutations are in the HEPN domain of theCRISPR enzyme or is a mutation as otherwise discussed herein. In someembodiments, the CRISPR enzyme has one or more mutations in a catalyticdomain, wherein when transcribed, the direct repeat sequence forms asingle stem loop and the guide sequence directs sequence-specificbinding of a CRISPR complex to the target sequence, and wherein theenzyme further comprises a functional domain. In some embodiments, thefunctional domain is a. In some embodiments, the functional domain is atranscription repression domain, preferably KRAB. In some embodiments,the transcription repression domain is SID, or concatemers of SID (egSID4X). In some embodiments, the functional domain is an epigeneticmodifying domain, such that an epigenetic modifying enzyme is provided.In some embodiments, the functional domain is an activation domain,which may be the P65 activation domain.

Delivery of the CRISPR Effector Protein Complex or Components Thereof

Through this disclosure and the knowledge in the art, TALEs, CRISPR-Cassystems, or components thereof or nucleic acid molecules thereof ornucleic acid molecules encoding or providing components thereof may bedelivered by a delivery system herein described both generally and indetail.

Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, forinstance a Type VI protein such as C2c2, and/or any of the present RNAs,for instance a guide RNA, can be delivered using any suitable vector,e.g., plasmid or viral vectors, such as adeno associated virus (AAV),lentivirus, adenovirus or other viral vector types, or combinationsthereof. Effector proteins and one or more guide RNAs can be packagedinto one or more vectors, e.g., plasmid or viral vectors. In someembodiments, the vector, e.g., plasmid or viral vector is delivered tothe tissue of interest by, for example, an intramuscular injection,while other times the delivery is via intravenous, transdermal,intranasal, oral, mucosal, or other delivery methods. Such delivery maybe either via a single dose, or multiple doses. One skilled in the artunderstands that the actual dosage to be delivered herein may varygreatly depending upon a variety of factors, such as the vector choice,the target cell, organism, or tissue, the general condition of thesubject to be treated, the degree of transformation/modification sought,the administration route, the administration mode, the type oftransformation/modification sought, etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N. J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×105 particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×106 particles(for example, about 1×106-1×1012 particles), more preferably at leastabout 1×107 particles, more preferably at least about 1×108 particles(e.g., about 1×108-1×1011 particles or about 1×108-1×1012 particles),and most preferably at least about 1×100 particles (e.g., about1×109-1×1010 particles or about 1×109-1×1012 particles), or even atleast about 1×1010 particles (e.g., about 1×1010-1×1012 particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×1014 particles, preferably no more than about 1×1013 particles,even more preferably no more than about 1×1012 particles, even morepreferably no more than about 1×1011 particles, and most preferably nomore than about 1×1010 particles (e.g., no more than about 1×109articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×106 particle units (pu), about 2×106 pu,about 4×106 pu, about 1×107 pu, about 2×107 pu, about 4×107 pu, about1×108 pu, about 2×108 pu, about 4×108 pu, about 1×109 pu, about 2×109pu, about 4×109 pu, about 1×1010 pu, about 2×1010 pu, about 4×1010 pu,about 1×1011 pu, about 2×1011 pu, about 4×1011 pu, about 1×1012 pu,about 2×1012 pu, or about 4×1012 pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×1010 to about 1×1010 functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×105 to 1×1050 genomes AAV,from about 1×108 to 1×1020 genomes AAV, from about 1×1010 to about1×1016 genomes, or about 1×1011 to about 1×1016 genomes AAV. A humandosage may be about 1×1013 genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding an nucleicacid-targeting CRISPR enzyme, operably linked to said promoter; (iii) aselectable marker; (iv) an origin of replication; and (v) atranscription terminator downstream of and operably linked to (ii). Theplasmid can also encode the RNA components of a CRISPR complex, but oneor more of these may instead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference. Delivery systemsaimed specifically at the enhanced and improved delivery of siRNA intomammalian cells have been developed, (see, for example, Shen et al FEBSLet. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010;Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol.Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 andSimeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to thepresent invention. siRNA has recently been successfully used forinhibition of gene expression in primates (see for example. Tolentino etal., Retina 24(4):660 which may also be applied to the presentinvention.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver nucleic acid-targeting Cas proteinCas9 and guideRNAgRNA (and, for instance, HR repair template) into cells usingliposomes or particles. Thus delivery of the nucleic acid-targeting Casprotein/CRISPR enzyme, such as a CasCas9 and/or delivery of the guideRNAs of the invention may be in RNA form and via microvesicles,liposomes or particles. For example, Cas mRNA and guide RNA can bepackaged into liposomal particles for delivery in vivo. Liposomaltransfection reagents such as lipofectamine from Life Technologies andother reagents on the market can effectively deliver RNA molecules intothe liver.

Means of delivery of RNA also preferred include delivery of RNA viananoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei,Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticlesfor small interfering RNA delivery to endothelial cells, AdvancedFunctional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A.,Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-basednanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267:9-21, 2010, PMID: 20059641). Indeed, exosomes have been shown to beparticularly useful in delivery siRNA, a system with some parallels tothe RNA-targeting system. For instance, El-Andaloussi S, et al.(“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc.2012 December; 7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012Nov. 15.) describe how exosomes are promising tools for drug deliveryacross different biological barriers and can be harnessed for deliveryof siRNA in vitro and in vivo. Their approach is to generate targetedexosomes through transfection of an expression vector, comprising anexosomal protein fused with a peptide ligand. The exosomes are thenpurified and characterized from transfected cell supernatant, then RNAis loaded into the exosomes. Delivery or administration according to theinvention can be performed with exosomes, in particular but not limitedto the brain. Vitamin E (α-tocopherol) may be conjugated with nucleicacid-targeting Cas protein and delivered to the brain along with highdensity lipoprotein (HDL), for example in a similar manner as was doneby Uno et al. (HUMAN GENE THERAPY 22:711-719 (June 2011)) for deliveringshort-interfering RNA (siRNA) to the brain. Mice were infused viaOsmotic minipumps (model 1007D; Alzet, Cupertino, Calif.) filled withphosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL andconnected with Brain Infusion Kit 3 (Alzet). A brain-infusion cannulawas placed about 0.5 mm posterior to the bregma at midline for infusioninto the dorsal third ventricle. Uno et al. found that as little as 3nmol of Toc-siRNA with HDL could induce a target reduction in comparabledegree by the same ICV infusion method. A similar dosage of nucleicacid-targeting effector protein conjugated to α-tocopherol andco-administered with HDL targeted to the brain may be contemplated forhumans in the present invention, for example, about 3 nmol to about 3μmol of nucleic acid-targeting effector protein targeted to the brainmay be contemplated. Zou et al. ((HUMAN GENE THERAPY 22:465-475 (April2011)) describes a method of lentiviral-mediated delivery ofshort-hairpin RNAs targeting PKCγ for in vivo gene silencing in thespinal cord of rats. Zou et al. administered about 10 μl of arecombinant lentivirus having a titer of 1×10⁹ transducing units (TU)/mlby an intrathecal catheter. A similar dosage of nucleic acid-targetingeffector protein expressed in a lentiviral vector targeted to the brainmay be contemplated for humans in the present invention, for example,about 10-50 ml of nucleic acid-targeting effector protein targeted tothe brain in a lentivirus having a titer of 1×10⁹ transducing units(TU)/ml may be contemplated.

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g., byinjection. Injection can be performed stereotactically via a craniotomy.

Packaging and Promoters Generally

Ways to package nucleic acid-targeting effector coding nucleic acidmolecules, e.g., DNA, into vectors, e.g., viral vectors, to mediategenome modification in vivo include:

To Achieve NHEJ-Mediated Gene Knockout:

Single Virus Vector:

Vector containing two or more expression cassettes:

Promoter-nucleic acid-targeting effector protein coding nucleic acidmolecule-terminator

Promoter-guide RNA1-terminator

Promoter-guide RNA (N)-terminator (up to size limit of vector)

Double Virus Vector:

Vector 1 containing one expression cassette for driving the expressionof nucleic acid-targeting effector protein

Promoter-nucleic acid-targeting effector protein coding nucleic acidmolecule-terminator

Vector 2 containing one more expression cassettes for driving theexpression of one or more guideRNAs

Promoter-guide RNA1-terminator

Promoter-guide RNA1 (N)-terminator (up to size limit of vector)

To Mediate Homology-Directed Repair.

In addition to the single and double virus vector approaches describedabove, an additional vector is used to deliver a homology-direct repairtemplate.

The promoter used to drive nucleic acid-targeting effector proteincoding nucleic acid molecule expression can include:

AAV ITR can serve as a promoter: this is advantageous for eliminatingthe need for an additional promoter element (which can take up space inthe vector). The additional space freed up can be used to drive theexpression of additional elements (gRNA, etc.). Also, ITR activity isrelatively weaker, so can be used to reduce potential toxicity due toover expression of nucleic acid-targeting effector protein.

For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40,Ferritin heavy or light chains, etc.

For brain or other CNS expression, can use promoters: SynapsinI for allneurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT forGABAergic neurons, etc.

For liver expression, can use Albumin promoter.

For lung expression, can use SP-B.

For endothelial cells, can use ICAM.

For hematopoietic cells can use IFNbeta or CD45.

For Osteoblasts can use OG-2.

The promoter used to drive guide RNA can include:

Pol III promoters such as U6 or H1

Use of Pol II promoter and intronic cassettes to express guide RNA

Adeno Associated Virus (AAV)

nucleic acid-targeting effector protein and one or more guide RNA can bedelivered using adeno associated virus (AAV), lentivirus, adenovirus orother plasmid or viral vector types, in particular, using formulationsand doses from, for example, U.S. Pat. No. 8,454,972 (formulations,doses for adenovirus), U.S. Pat. No. 8,404,658 (formulations, doses forAAV) and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids)and from clinical trials and publications regarding the clinical trialsinvolving lentivirus, AAV and adenovirus. For examples, for AAV, theroute of administration, formulation and dose can be as in U.S. Pat. No.8,454,972 and as in clinical trials involving AAV. For Adenovirus, theroute of administration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g., a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specificgenome/transcriptome modification, the expression of nucleicacid-targeting effector protein can be driven by a cell-type specificpromoter. For example, liver-specific expression might use the Albuminpromoter and neuron-specific expression (e.g., for targeting CNSdisorders) might use the Synapsin I promoter.

In terms of in vivo delivery, AAV is advantageous over other viralvectors for a couple of reasons:

-   -   Low toxicity (this may be due to the purification method not        requiring ultra centrifugation of cell particles that can        activate the immune response) and    -   Low probability of causing insertional mutagenesis because it        doesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that nucleicacid-targeting effector protein (such as a Type VI protein such as C2c2)as well as a promoter and transcription terminator have to be all fitinto the same viral vector. Therefore embodiments of the inventioninclude utilizing homologs of nucleic acid-targeting effector proteinthat are shorter.

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV of the AAV with regard to the cells to betargeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsidAAV1, AAV2, AAV5 or any combination thereof for targeting brain orneuronal cells; and one can select AAV4 for targeting cardiac tissue.AAV8 is useful for delivery to the liver. The herein promoters andvectors are preferred individually. A tabulation of certain AAVserotypes as to these cells (see Grimm, D. et al, J. Virol. 82:5887-5911 (2008)) is as follows:

TABLE 10 Cell AAV- AAV- AAV- AAV- AAV- AAV- AAV- AAV- Line 1 2 3 4 5 6 89 Huh-7  13 100 2.5 0.0 0.1 10 0.7 0.0 HEK293  25 100 2.5 0.1 0.1 5 0.70.1 HeLa   3 100 2.0 0.1 6.7 1 0.2 0.1 HepG2   3 100 16.7 0.3 1.7 5 0.3ND Hep1A  20 100 0.2 1.0 0.1 1 0.2 0.0 911  17 100 11 0.2 0.1 17 0.1 NDCHO  100 100 14 1.4 333 50 10 1.0 COS  33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.0 0.2 NIH3T3  10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1 HT1180  20 100 10 0.1 0.3 33 0.5 0.1 Mono-1111 100 ND ND 125 1429 ND ND cytes Imma- 2500 100 ND ND 222 2857 ND NDture DC Mature 2222 100 ND ND 333 3333 ND ND DC

Lentivirus

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types.

Lentiviruses may be prepared as follows. After cloning pCasES10 (whichcontains a lentiviral transfer plasmid backbone), HEK293FT at lowpassage (p=5) were seeded in a T-75 flask to 50% confluence the daybefore transfection in DMEM with 10% fetal bovine serum and withoutantibiotics. After 20 hours, media was changed to OptiMEM (serum-free)media and transfection was done 4 hours later. Cells were transfectedwith 10 μg of lentiviral transfer plasmid (pCasES10) and the followingpackaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 ug ofpsPAX2 (gag/pol/rev/tat). Transfection was done in 4 mL OptiMEM with acationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul Plusreagent). After 6 hours, the media was changed to antibiotic-free DMEMwith 10% fetal bovine serum. These methods use serum during cellculture, but serum-free methods are preferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 um low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 ul of DMEM overnight at 4 C. They were then aliquottedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinffctious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the nucleic acid-targeting system of thepresent invention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used/and or adapted to the nucleic acid-targeting system of thepresent invention. A minimum of 2.5×106 CD34+ cells per kilogram patientweight may be collected and prestimulated for 16 to 20 hours in X-VIVO15 medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×106 cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm2 tissue culture flasks coated with fibronectin (25mg/cm2) (RetroNectin, Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571; US20110293571, US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015.

RNA Delivery

RNA delivery: The nucleic acid-targeting Cas protein, for instance aType VI protein such as C2c2, and/or guide RNA, can also be delivered inthe form of RNA. nucleic acid-targeting Cas protein (such as a Type VIprotein such as C2c2) mRNA can be generated using in vitrotranscription. For example, nucleic acid-targeting effector protein(such as a Type VI protein such as C2c2) mRNA can be synthesized using aPCR cassette containing the following elements: T7_promoter-kozaksequence (GCCACC)-effector protrein-3′ UTR from beta globin-polyA tail(a string of 120 or more adenines). The cassette can be used fortranscription by T7 polymerase. Guide RNAs can also be transcribed usingin vitro transcription from a cassette containing T7_promoter-GG-guideRNA sequence.

To enhance expression and reduce possible toxicity, the nucleicacid-targeting effector protein-coding sequence and/or the guide RNA canbe modified to include one or more modified nucleoside e.g., usingpseudo-U or 5-Methyl-C.

mRNA delivery methods are especially promising for liver deliverycurrently.

Much clinical work on RNA delivery has focused on RNAi or antisense, butthese systems can be adapted for delivery of RNA for implementing thepresent invention. References below to RNAi etc. should be readaccordingly.

Particle Delivery Systems and/or Formulations:

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter. Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.diameter) of less than 100 microns (m). In some embodiments, inventiveparticles have a greatest dimension of less than 10 m. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarisation interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of CRISPR-Cassystem e.g., CRISPR enzyme or mRNA or guide RNA, or any combinationthereof, and may include additional carriers and/or excipients) toprovide particles of an optimal size for delivery for any in vitro, exvivo and/or in vivo application of the present invention. In certainpreferred embodiments, particle dimension (e.g., diameter)characterization is based on measurements using dynamic laser scattering(DLS). Mention is made of U.S. Pat. Nos. 8,709,843; 6,007,845;5,855,913; 5,985,309; 5,543,158; and the publication by James E. Dahlmanand Carmen Barnes et al. Nature Nanotechnology (2014) published online11 May 2014, doi:10.1038/nnano.2014.84, concerning particles, methods ofmaking and using them and measurements thereof.

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to solid, semi-solid,emulsion, or colloidal particles. As such any of the delivery systemsdescribed herein, including but not limited to, e.g., lipid-basedsystems, liposomes, micelles, microvesicles, exosomes, or gene gun maybe provided as particle delivery systems within the scope of the presentinvention.

Particles

CRISPR enzyme mRNA and guide RNA may be delivered simultaneously usingparticles or lipid envelopes; for instance, CRISPR enzyme and RNA of theinvention, e.g., as a complex, can be delivered via a particle as inDahlman et al., WO2015089419 A2 and documents cited therein, such as 7C1(see, e.g., James E. Dahlman and Carmen Barnes et al. NatureNanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84), e.g., delivery particle comprising lipid orlipidoid and hydrophilic polymer, e.g., cationic lipid and hydrophilicpolymer, for instance wherein the the cationic lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or whereinthe hydrophilic polymer comprises ethylene glycol or polyethylene glycol(PEG); and/or wherein the particle further comprises cholesterol (e.g.,particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0;formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0;formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5), whereinparticles are formed using an efficient, multistep process whereinfirst, effector protein and RNA are mixed together, e.g., at a 1:1 molarratio, e.g., at room temperature, e.g., for 30 minutes, e.g., insterile, nuclease free 1×PBS; and separately, DOTAP, DMPC, PEG, andcholesterol as applicable for the formulation are dissolved in alcohol,e.g., 100% ethanol; and, the two solutions are mixed together to formparticles containing the complexes).

Nucleic acid-targeting effector proteins (such as a Type VI protein suchas C2c2) mRNA and guide RNA may be delivered simultaneously usingparticles or lipid envelopes.

For example, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and invivo mRNA delivery using lipid-enveloped pH-responsive polymernanoparticles” Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable core-shellstructured particles with a poly(β-amino ester) (PBAE) core enveloped bya phospholipid bilayer shell. These were developed for in vivo mRNAdelivery. The pH-responsive PBAE component was chosen to promoteendosome disruption, while the lipid surface layer was selected tominimize toxicity of the polycation core. Such are, therefore, preferredfor delivering RNA of the present invention.

In one embodiment, particles based on self-assembling bioadhesivepolymers are contemplated, which may be applied to oral delivery ofpeptides, intravenous delivery of peptides and nasal delivery ofpeptides, all to the brain. Other embodiments, such as oral absorptionand ocular delivery of hydrophobic drugs are also contemplated. Themolecular envelope technology involves an engineered polymer envelopewhich is protected and delivered to the site of the disease (see, e.g.,Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026; Siew, A., et al. MolPharm, 2012. 9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012.161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012. 9(6):1665-80;Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N. L., etal. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N. L., et al. J RamanSpect, 2012. 43(5):681-688; Ahmad, S., et al. J Royal Soc Interface2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9 andUchegbu, I. F., et al. Int J Pharm, 2001. 224:185-199). Doses of about 5mg/kg are contemplated, with single or multiple doses, depending on thetarget tissue.

In one embodiment, particles that can deliver RNA to a cancer cell tostop tumor growth developed by Dan Anderson's lab at MIT may be used/andor adapted to the nucleic acid-targeting system of the presentinvention. In particular, the Anderson lab developed fully automated,combinatorial systems for the synthesis, purification, characterization,and formulation of new biomaterials and nanoformulations. See, e.g.,Alabi et al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110(32):12881-6;Zhang et al., Adv Mater. 2013 Sep. 6; 25(33):4641-5; Jiang et al., NanoLett. 2013 Mar. 13; 13(3): 1059-64; Karagiannis et al., ACS Nano. 2012Oct. 23; 6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28;6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93.

US patent application 20110293703 relates to lipidoid compounds are alsoparticularly useful in the administration of polynucleotides, which maybe applied to deliver the nucleic acid-targeting system of the presentinvention. In one aspect, the aminoalcohol lipidoid compounds arecombined with an agent to be delivered to a cell or a subject to formmicroparticles, nanoparticles, liposomes, or micelles. The agent to bedelivered by the particles, liposomes, or micelles may be in the form ofa gas, liquid, or solid, and the agent may be a polynucleotide, protein,peptide, or small molecule. The minoalcohol lipidoid compounds may becombined with other aminoalcohol lipidoid compounds, polymers (syntheticor natural), surfactants, cholesterol, carbohydrates, proteins, lipids,etc. to form the particles. These particles may then optionally becombined with a pharmaceutical excipient to form a pharmaceuticalcomposition.

US Patent Publication No. 20110293703 also provides methods of preparingthe aminoalcohol lipidoid compounds. One or more equivalents of an amineare allowed to react with one or more equivalents of anepoxide-terminated compound under suitable conditions to form anaminoalcohol lipidoid compound of the present invention. In certainembodiments, all the amino groups of the amine are fully reacted withthe epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30-100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell.

US Patent Publication No. 20130302401 relates to a class ofpoly(beta-amino alcohols) (PBAAs) has been prepared using combinatorialpolymerization. The inventive PBAAs may be used in biotechnology andbiomedical applications as coatings (such as coatings of films ormultilayer films for medical devices or implants), additives, materials,excipients, non-biofouling agents, micropatterning agents, and cellularencapsulation agents. When used as surface coatings, these PBAAselicited different levels of inflammation, both in vitro and in vivo,depending on their chemical structures. The large chemical diversity ofthis class of materials allowed us to identify polymer coatings thatinhibit macrophage activation in vitro. Furthermore, these coatingsreduce the recruitment of inflammatory cells, and reduce fibrosis,following the subcutaneous implantation of carboxylated polystyrenemicroparticles. These polymers may be used to form polyelectrolytecomplex capsules for cell encapsulation. The invention may also havemany other biological applications such as antimicrobial coatings, DNAor siRNA delivery, and stem cell tissue engineering. The teachings of USPatent Publication No. 20130302401 may be applied to the nucleicacid-targeting system of the present invention.

In another embodiment, lipid nanoparticles (LNPs) are contemplated. Anantitransthyretin small interfering RNA has been encapsulated in lipidnanoparticles and delivered to humans (see, e.g., Coelho et al., N EnglJ Med 2013; 369:819-29), and such a system may be adapted and applied tothe nucleic acid-targeting system of the present invention. Doses ofabout 0.01 to about 1 mg per kg of body weight administeredintravenously are contemplated. Medications to reduce the risk ofinfusion-related reactions are contemplated, such as dexamethasone,acetampinophen, diphenhydramine or cetirizine, and ranitidine arecontemplated. Multiple doses of about 0.3 mg per kilogram every 4 weeksfor five doses are also contemplated.

LNPs have been shown to be highly effective in delivering siRNAs to theliver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No. 4, pages 363-470) and are therefore contemplated for deliveringRNA encoding nucleic acid-targeting effector protein to the liver. Adosage of about four doses of 6 mg/kg of the LNP every two weeks may becontemplated. Tabernero et al. demonstrated that tumor regression wasobserved after the first 2 cycles of LNPs dosed at 0.7 mg/kg, and by theend of 6 cycles the patient had achieved a partial response withcomplete regression of the lymph node metastasis and substantialshrinkage of the liver tumors. A complete response was obtained after 40doses in this patient, who has remained in remission and completedtreatment after receiving doses over 26 months. Two patients with RCCand extrahepatic sites of disease including kidney, lung, and lymphnodes that were progressing following prior therapy with VEGF pathwayinhibitors had stable disease at all sites for approximately 8 to 12months, and a patient with PNET and liver metastases continued on theextension study for 18 months (36 doses) with stable disease.

However, the charge of the LNP must be taken into consideration. Ascationic lipids combined with negatively charged lipids to inducenonbilayer structures that facilitate intracellular delivery. Becausecharged LNPs are rapidly cleared from circulation following intravenousinjection, ionizable cationic lipids with pKa values below 7 weredeveloped (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12,pages 1286-2200, December 2011). Negatively charged polymers such as RNAmay be loaded into LNPs at low pH values (e.g., pH 4) where theionizable lipids display a positive charge. However, at physiological pHvalues, the LNPs exhibit a low surface charge compatible with longercirculation times. Four species of ionizable cationic lipids have beenfocused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).It has been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, December 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR-Cas encapsulation may be used/and oradapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(co-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificnucleic acid-targeting complex (CRISPR-Cas) RNA may be encapsulated inLNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationiclipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios).When required, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) may beincorporated to assess cellular uptake, intracellular delivery, andbiodistribution. Encapsulation may be performed by dissolving lipidmixtures comprised of cationic lipid:DSPC:cholesterol:PEG-c-DOMG(40:10:40:10 molar ratio) in ethanol to a final lipid concentration of10 mmol/l. This ethanol solution of lipid may be added drop-wise to 50mmol/l citrate, pH 4.0 to form multilamellar vesicles to produce a finalconcentration of 30% ethanol vol/vol. Large unilamellar vesicles may beformed following extrusion of multilamellar vesicles through two stacked80 nm Nuclepore polycarbonate filters using the Extruder (NorthernLipids, Vancouver, Canada). Encapsulation may be achieved by adding RNAdissolved at 2 mg/ml in 50 mmol/l citrate, pH 4.0 containing 30% ethanolvol/vol drop-wise to extruded preformed large unilamellar vesicles andincubation at 31° C. for 30 minutes with constant mixing to a finalRNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol andneutralization of formulation buffer were performed by dialysis againstphosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2regenerated cellulose dialysis membranes. Particle size distribution maybe determined by dynamic light scattering using a NICOMP 370 particlesizer, the vesicle/intensity modes, and Gaussian fitting (NicompParticle Sizing, Santa Barbara, Calif.). The particle size for all threeLNP systems may be ˜70 nm in diameter. RNA encapsulation efficiency maybe determined by removal of free RNA using VivaPureD MiniH columns(Sartorius Stedim Biotech) from samples collected before and afterdialysis. The encapsulated RNA may be extracted from the elutedparticles and quantified at 260 nm. RNA to lipid ratio was determined bymeasurement of cholesterol content in vesicles using the Cholesterol Eenzymatic assay from Wako Chemicals USA (Richmond, Va.). In conjunctionwith the herein discussion of LNPs and PEG lipids, PEGylated liposomesor LNPs are likewise suitable for delivery of a nucleic acid-targetingsystem or components thereof.

Preparation of large LNPs may be used/and or adapted from Rosin et al,Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011. Alipid premix solution (20.4 mg/ml total lipid concentration) may beprepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premixat a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids maybe subsequently hydrated by combining the mixture with 1.85 volumes ofcitrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting inspontaneous liposome formation in aqueous buffer containing 35% ethanol.The liposome solution may be incubated at 37° C. to allow fortime-dependent increase in particle size. Aliquots may be removed atvarious times during incubation to investigate changes in liposome sizeby dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments,Worcestershire, UK). Once the desired particle size is achieved, anaqueous PEG lipid solution (stock=10 mg/ml PEG-DMG in 35% (vol/vol)ethanol) may be added to the liposome mixture to yield a final PEG molarconcentration of 3.5% of total lipid. Upon addition of PEG-lipids, theliposomes should their size, effectively quenching further growth. RNAmay then be added to the empty liposomes at a RNA to total lipid ratioof approximately 1:10 (wt:wt), followed by incubation for 30 minutes at37° C. to form loaded LNPs. The mixture may be subsequently dialyzedovernight in PBS and filtered with a 0.45-μm syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other particles(particularly gold particles) are also contemplated as a means todelivery nucleic acid-targeting system to intended targets. Significantdata show that AuraSense Therapeutics' Spherical Nucleic Acid (SNA™)constructs, based upon nucleic acid-functionalized gold particles, areuseful.

Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen etal., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling particles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGF R2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of nucleic acid-targeting complex RNA isenvisioned for delivery in the self-assembling particles of Schiffelerset al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no.39) may also be applied to the present invention. The nanoplexes ofBartlett et al. are prepared by mixing equal volumes of aqueoussolutions of cationic polymer and nucleic acid to give a net molarexcess of ionizable nitrogen (polymer) to phosphate (nucleic acid) overthe range of 2 to 6. The electrostatic interactions between cationicpolymers and nucleic acid resulted in the formation of polyplexes withaverage particle size distribution of about 100 nm, hence referred tohere as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered fromMacrocyclics (Dallas, Tex.). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNAsense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove tracemetal contaminants. Tf-targeted and nontargeted siRNA particles may beformed by using cyclodextrin-containing polycations. Typically,particles were formed in water at a charge ratio of 3 (+/−) and an siRNAconcentration of 0.5 g/liter. One percent of the adamantane-PEGmolecules on the surface of the targeted particles were modified with Tf(adamantane-PEG-Tf). The particles were suspended in a 5% (wt/vol)glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA clinicaltrial that uses a targeted particle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetedparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The particles comprise, consist essentially of, orconsist of a synthetic delivery system containing: (1) a linear,cyclodextrin-based polymer (CDP), (2) a human transferrin protein (TF)targeting ligand displayed on the exterior of the nanoparticle to engageTF receptors (TFR) on the surface of the cancer cells, (3) a hydrophilicpolymer (polyethylene glycol (PEG) used to promote nanoparticlestability in biological fluids), and (4) siRNA designed to reduce theexpression of the RRM2 (sequence used in the clinic was previouslydenoted siR2B+5). The TFR has long been known to be upregulated inmalignant cells, and RRM2 is an established anti-cancer target. Theseparticles (clinical version denoted as CALAA-01) have been shown to bewell tolerated in multi-dosing studies in non-human primates. Although asingle patient with chronic myeloid leukaemia has been administeredsiRNA by liposomal delivery, Davis et al.'s clinical trial is theinitial human trial to systemically deliver siRNA with a targeteddelivery system and to treat patients with solid cancer. To ascertainwhether the targeted delivery system can provide effective delivery offunctional siRNA to human tumours, Davis et al. investigated biopsiesfrom three patients from three different dosing cohorts; patients A, Band C, all of whom had metastatic melanoma and received CALAA-01 dosesof 18, 24 and 30 mg m-2 siRNA, respectively. Similar doses may also becontemplated for the nucleic acid-targeting system of the presentinvention. The delivery of the invention may be achieved with particlescontaining a linear, cyclodextrin-based polymer (CDP), a humantransferrin protein (TF) targeting ligand displayed on the exterior ofthe particle to engage TF receptors (TFR) on the surface of the cancercells and/or a hydrophilic polymer (for example, polyethylene glycol(PEG) used to promote particle stability in biological fluids).

In terms of this invention, it is preferred to have one or morecomponents of nucleic acid-targeting complex, e.g., nucleicacid-targeting effector protein or mRNA, or guide RNA delivered usingparticles or lipid envelopes. Other delivery systems or vectors are maybe used in conjunction with the particle aspects of the invention.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In certain preferred embodiments, nanoparticles ofthe invention have a greatest dimension (e.g., diameter) of 500 nm orless. In other preferred embodiments, nanoparticles of the inventionhave a greatest dimension ranging between 25 nm and 200 nm. In otherpreferred embodiments, particles of the invention have a greatestdimension of 100 nm or less. In other preferred embodiments,nanoparticles of the invention have a greatest dimension ranging between35 nm and 60 nm.

Particles encompassed in the present invention may be provided indifferent forms, e.g., as solid particles (e.g., metal such as silver,gold, iron, titanium), non-metal, lipid-based solids, polymers),suspensions of particles, or combinations thereof. Metal, dielectric,and semiconductor particles may be prepared, as well as hybridstructures (e.g., core-shell particles). Particles made ofsemiconducting material may also be labeled quantum dots if they aresmall enough (typically sub 10 nm) that quantization of electronicenergy levels occurs. Such nanoscale particles are used in biomedicalapplications as drug carriers or imaging agents and may be adapted forsimilar purposes in the present invention.

Semi-solid and soft particles have been manufactured, and are within thescope of the present invention. A prototype particle of semi-solidnature is the liposome. Various types of liposome particles arecurrently used clinically as delivery systems for anticancer drugs andvaccines. Particles with one half hydrophilic and the other halfhydrophobic are termed Janus particles and are particularly effectivefor stabilizing emulsions. They can self-assemble at water/oilinterfaces and act as solid surfactants.

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingpolymer conjugated to a surfactant, hydrophilic polymer or lipid.

U.S. Pat. No. 6,007,845, incorporated herein by reference, providesparticles which have a core of a multiblock copolymer formed bycovalently linking a multifunctional compound with one or morehydrophobic polymers and one or more hydrophilic polymers, and contain abiologically active material.

U.S. Pat. No. 5,855,913, incorporated herein by reference, provides aparticulate composition having aerodynamically light particles having atap density of less than 0.4 g/cm3 with a mean diameter of between 5 μmand 30 μm, incorporating a surfactant on the surface thereof for drugdelivery to the pulmonary system.

U.S. Pat. No. 5,985,309, incorporated herein by reference, providesparticles incorporating a surfactant and/or a hydrophilic or hydrophobiccomplex of a positively or negatively charged therapeutic or diagnosticagent and a charged molecule of opposite charge for delivery to thepulmonary system.

U.S. Pat. No. 5,543,158, incorporated herein by reference, providesbiodegradable injectable particles having a biodegradable solid corecontaining a biologically active material and poly(alkylene glycol)moieties on the surface.

WO2012135025 (also published as US20120251560), incorporated herein byreference, describes conjugated polyethyleneimine (PEI) polymers andconjugated aza-macrocycles (collectively referred to as “conjugatedlipomer” or “lipomers”). In certain embodiments, it can be envisionedthat such methods and materials of herein-cited documents, e.g.,conjugated lipomers can be used in the context of the nucleicacid-targeting system to achieve in vitro, ex vivo and in vivo genomicperturbations to modify gene expression, including modulation of proteinexpression.

In one embodiment, the particle may be epoxide-modified lipid-polymer,advantageously 7C1 (see, e.g., James E. Dahlman and Carmen Barnes et al.Nature Nanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84). C71 was synthesized by reacting C15epoxide-terminated lipids with PEI600 at a 14:1 molar ratio, and wasformulated with C14PEG2000 to produce particles (diameter between 35 and60 nm) that were stable in PBS solution for at least 40 days.

An epoxide-modified lipid-polymer may be utilized to deliver the nucleicacid-targeting system of the present invention to pulmonary,cardiovascular or renal cells, however, one of skill in the art mayadapt the system to deliver to other target organs. Dosage ranging fromabout 0.05 to about 0.6 mg/kg are envisioned. Dosages over several daysor weeks are also envisioned, with a total dosage of about 2 mg/kg.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (MHC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by particle tracking analysis (NTA) andelectron microscopy. Alvarez-Erviti et al. obtained 6-12 μg of exosomes(measured based on protein concentration) per 106 cells.

Next, Alvarez-Erviti et al. investigated the possibility of loadingmodified exosomes with exogenous cargoes using electroporation protocolsadapted for nanoscale applications. As electroporation for membraneparticles at the nanometer scale is not well-characterized, nonspecificCy5-labeled RNA was used for the empirical optimization of theelectroporation protocol. The amount of encapsulated RNA was assayedafter ultracentrifugation and lysis of exosomes. Electroporation at 400V and 125 μF resulted in the greatest retention of RNA and was used forall subsequent experiments.

Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNAencapsulated in 150 μg of RVG exosomes to normal C57BL/6 mice andcompared the knockdown efficiency to four controls: untreated mice, miceinjected with RVG exosomes only, mice injected with BACE1 siRNAcomplexed to an in vivo cationic liposome reagent and mice injected withBACE1 siRNA complexed to RVG-9R, the RVG peptide conjugated to 9D-arginines that electrostatically binds to the siRNA. Cortical tissuesamples were analyzed 3 d after administration and a significant proteinknockdown (45%, P<0.05, versus 62%, P<0.01) in both siRNA-RVG-9R-treatedand siRNARVG exosome-treated mice was observed, resulting from asignificant decrease in BACE1 mRNA levels (66% [+ or -] 15%, P<0.001 and61% [+ or -] 13% respectively, P<0.01). Moreover, Applicantsdemonstrated a significant decrease (55%, P<0.05) in the total[beta]-amyloid 1-42 levels, a main component of the amyloid plaques inAlzheimer's pathology, in the RVG-exosome-treated animals. The decreaseobserved was greater than the β-amyloid 1-40 decrease demonstrated innormal mice after intraventricular injection of BACE1 inhibitors.Alvarez-Erviti et al. carried out 5′-rapid amplification of cDNA ends(RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA.

Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomesinduced immune responses in vivo by assessing IL-6, IP-10, TNFα andIFN-α serum concentrations. Following exosome treatment, nonsignificantchanges in all cytokines were registered similar to siRNA-transfectionreagent treatment in contrast to siRNA-RVG-9R, which potently stimulatedIL-6 secretion, confirming the immunologically inert profile of theexosome treatment. Given that exosomes encapsulate only 20% of siRNA,delivery with RVG-exosome appears to be more efficient than RVG-9Rdelivery as comparable mRNA knockdown and greater protein knockdown wasachieved with fivefold less siRNA without the corresponding level ofimmune stimulation. This experiment demonstrated the therapeuticpotential of RVG-exosome technology, which is potentially suited forlong-term silencing of genes related to neurodegenerative diseases. Theexosome delivery system of Alvarez-Erviti et al. may be applied todeliver the nucleic acid-targeting system of the present invention totherapeutic targets, especially neurodegenerative diseases. A dosage ofabout 100 to 1000 mg of nucleic acid-targeting system encapsulated inabout 100 to 1000 mg of RVG exosomes may be contemplated for the presentinvention.

El-Andaloussi et al. (Nature Protocols 7, 2112-2126(2012)) discloses howexosomes derived from cultured cells can be harnessed for delivery ofRNA in vitro and in vivo. This protocol first describes the generationof targeted exosomes through transfection of an expression vector,comprising an exosomal protein fused with a peptide ligand. Next,El-Andaloussi et al. explain how to purify and characterize exosomesfrom transfected cell supernatant. Next, El-Andaloussi et al. detailcrucial steps for loading RNA into exosomes. Finally, El-Andaloussi etal. outline how to use exosomes to efficiently deliver RNA in vitro andin vivo in mouse brain. Examples of anticipated results in whichexosome-mediated RNA delivery is evaluated by functional assays andimaging are also provided. The entire protocol takes ˜3 weeks. Deliveryor administration according to the invention may be performed usingexosomes produced from self-derived dendritic cells. From the hereinteachings, this can be employed in the practice of the invention

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention.

Exosomes from plasma can be prepared by centrifugation of buffy coat at900 g for 20 min to isolate the plasma followed by harvesting cellsupernatants, centrifuging at 300 g for 10 min to eliminate cells and at16 500 g for 30 min followed by filtration through a 0.22 mm filter.Exosomes are pelleted by ultracentrifugation at 120 000 g for 70 min.Chemical transfection of siRNA into exosomes is carried out according tothe manufacturer's instructions in RNAi Human/Mouse Starter Kit(Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a finalconcentration of 2 mmol/ml. After adding HiPerFect transfection reagent,the mixture is incubated for 10 min at RT. In order to remove the excessof micelles, the exosomes are re-isolated using aldehyde/sulfate latexbeads. The chemical transfection of nucleic acid-targeting system intoexosomes may be conducted similarly to siRNA. The exosomes may beco-cultured with monocytes and lymphocytes isolated from the peripheralblood of healthy donors. Therefore, it may be contemplated that exosomescontaining nucleic acid-targeting system may be introduced to monocytesand lymphocytes of and autologously reintroduced into a human.Accordingly, delivery or administration according to the invention maybe performed using plasma exosomes.

Liposomes

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review).

Liposomes can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes as drugcarriers. Although liposome formation is spontaneous when a lipid filmis mixed with an aqueous solution, it can also be expedited by applyingforce in the form of shaking by using a homogenizer, sonicator, or anextrusion apparatus (see, e.g., Spuch and Navarro, Journal of DrugDelivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155/2011/469679for review).

A liposome formulation may be mainly comprised of natural phospholipidsand lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline(DSPC), sphingomyelin, egg phosphatidylcholines andmonosialoganglioside. Since this formulation is made up of phospholipidsonly, liposomal formulations have encountered many challenges, one ofthe ones being the instability in plasma. Several attempts to overcomethese challenges have been made, specifically in the manipulation of thelipid membrane. One of these attempts focused on the manipulation ofcholesterol. Addition of cholesterol to conventional formulationsreduces rapid release of the encapsulated bioactive compound into theplasma or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increasesthe stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

In a particularly advantageous embodiment, Trojan Horse liposomes (alsoknown as Molecular Trojan Horses) are desirable and protocols may befound at cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long. Theseparticles allow delivery of a transgene to the entire brain after anintravascular injection. Without being bound by limitation, it isbelieved that neutral lipid particles with specific antibodiesconjugated to surface allow crossing of the blood brain barrier viaendocytosis. Applicant postulates utilizing Trojan Horse Liposomes todeliver the CRISPR family of nucleases to the brain via an intravascularinjection, which would allow whole brain transgenic animals without theneed for embryonic manipulation. About 1-5 g of DNA or RNA may becontemplated for in vivo administration in liposomes.

In another embodiment, the nucleic acid-targeting system or conmponentsthereof may be administered in liposomes, such as a stablenucleic-acid-lipid particle (SNALP) (see, e.g., Morrissey et al., NatureBiotechnology, Vol. 23, No. 8, August 2005). Daily intravenousinjections of about 1, 3 or 5 mg/kg/day of a specific nucleicacid-targeting system targeted in a SNALP are contemplated. The dailytreatment may be over about three days and then weekly for about fiveweeks. In another embodiment, a specific nucleic acid-targeting systemencapsulated SNALP) administered by intravenous injection to at doses ofabout 1 or 2.5 mg/kg are also contemplated (see, e.g., Zimmerman et al.,Nature Letters, Vol. 441, 4 May 2006). The SNALP formulation may containthe lipids 3-N-[(wmethoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic-acid-lipid particles (SNALPs) haveproven to be effective delivery molecules to highly vascularizedHepG2-derived liver tumors but not in poorly vascularized HCT-116derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780).The SNALP liposomes may be prepared by formulating D-Lin-DMA andPEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol andsiRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio ofCholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes areabout 80-100 nm in size.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine(Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, andcationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g.,Geisbert et al., Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kgtotal nucleic acid-targeting systemper dose administered as, forexample, a bolus intravenous infusion may be contemplated.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC;Avanti Polar Lipids Inc.), PEG-cDMA, and1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g.,Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for invivo studies may comprise a final lipid/RNA mass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids—an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties.

To date, two clinical programs have been initiated using SNALPformulations with RNA. Tekmira Pharmaceuticals recently completed aphase I single-dose study of SNALP-ApoB in adult volunteers withelevated LDL cholesterol. ApoB is predominantly expressed in the liverand jejunum and is essential for the assembly and secretion of VLDL andLDL. Seventeen subjects received a single dose of SNALP-ApoB (doseescalation across 7 dose levels). There was no evidence of livertoxicity (anticipated as the potential dose-limiting toxicity based onpreclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial.

Alnylam Pharmaceuticals has similarly advanced ALN-TTR01, which employsthe SNALP technology described above and targets hepatocyte productionof both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). ThreeATTR syndromes have been described: familial amyloidotic polyneuropathy(FAP) and familial amyloidotic cardiomyopathy (FAC)-both caused byautosomal dominant mutations in TTR; and senile systemic amyloidosis(SSA) cause by wildtype TTR. A placebo-controlled, singledose-escalation phase I trial of ALN-TTR01 was recently completed inpatients with ATTR. ALN-TTR01 was administered as a 15-minute IVinfusion to 31 patients (23 with study drug and 8 with placebo) within adose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatment was welltolerated with no significant increases in liver function tests.Infusion-related reactions were noted in 3 of 23 patients at ≥0.4 mg/kg;all responded to slowing of the infusion rate and all continued onstudy. Minimal and transient elevations of serum cytokines IL-6, IP-10and IL-1ra were noted in two patients at the highest dose of 1 mg/kg (asanticipated from preclinical and NHP studies). Lowering of serum TTR,the expected pharmacodynamics effect of ALN-TTR01, was observed at 1mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Niotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177). Thelipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) withmixing to a final ethanol and lipid concentration of 30% (vol/vol) and6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Northern Lipids) until a vesicle diameter of 70-90 nm, as determined bydynamic light scattering analysis, was obtained. This generally required1-3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueoussolution containing 30% ethanol) was added to the pre-equilibrated (35°C.) vesicles at a rate of ˜5 ml/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubatedfor a further 30 min at 35° C. to allow vesicle reorganization andencapsulation of the siRNA. The ethanol was then removed and theexternal buffer replaced with PBS (155 mM NaCl, 3 mM Na₂HPO₄, 1 mMKH₂PO₄, pH 7.5) by either dialysis or tangential flow diafiltration.siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti PolarLipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molarratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles,SNALP were dialyzed against PBS and filter sterilized through a 0.2 μmfilter before use. Mean particle sizes were 75-85 nm and 90-95% of thesiRNA was encapsulated within the lipid particles. The final siRNA/lipidratio in formulations used for in vivo testing was ˜0.15 (wt/wt).LNP-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile PBS immediately before use and theformulations were administered intravenously through the lateral tailvein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the nucleic acid-targeting system of thepresent invention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate nucleic acid-targeting system or componentsthereof or nucleic acid molecule(s) coding therefor e.g., similar toSiRNA (see, e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533),and hence may be employed in the practice of the invention. A preformedvesicle with the following lipid composition may be contemplated: aminolipid, distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11+0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the guide RNA. Particles containing the highly potentamino lipid 16 may be used, in which the molar ratio of the four lipidcomponents 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) whichmay be further optimized to enhance in vivo activity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume: 29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with the nucleicacid-targeting system of the present invention or component(s) thereofor nucleic acid molecule(s) coding therefor to form lipid nanoparticles(LNPs). Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG maybe formulated with RNA-targeting system instead of siRNA (see, e.g.,Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4;doi:10.1038/mtna.2011.3) using a spontaneous vesicle formationprocedure. The component molar ratio may be about 50/10/38.5/1.5(DLin-KC2-DMA or C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG). The final lipid:siRNA weight ratio may be˜12:1 and 9:1 in the case of DLin-KC2-DMA and C12-200 lipid particles(LNPs), respectively. The formulations may have mean particle diametersof ˜80 nm with >90% entrapment efficiency. A 3 mg/kg dose may becontemplated.

Tekmira has a portfolio of approximately 95 patent families, in the U.S.and abroad, that are directed to various aspects of LNPs and LNPformulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069;8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263;7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos 1766035;1519714; 1781593 and 1664316), all of which may be used and/or adaptedto the present invention.

The nucleic acid-targetingsystem or components thereof or nucleic acidmolecule(s) coding therefor may be delivered encapsulated in PLGAMicrospheres such as that further described in US published applications20130252281 and 20130245107 and 20130244279 (assigned to ModernaTherapeutics) which relate to aspects of formulation of compositionscomprising modified nucleic acid molecules which may encode a protein, aprotein precursor, or a partially or fully processed form of the proteinor a protein precursor. The formulation may have a molar ratio50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEGlipid). The PEG lipid may be selected from, but is not limited toPEG-c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC. See also, Schrumet al., Delivery and Formulation of Engineered Nucleic Acids, USpublished application 20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

US Patent Publication No. 20050019923 describes cationic dendrimers fordelivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step-wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesized from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropylenimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropylenimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine. andphosphorous containing compounds with a mixture of amine/amide orN—P(O₂)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropylenimine dendrimersfor gene delivery. Polypropylenimine dendrimers have also been studiedas pH sensitive controlled release systems for drug delivery and fortheir encapsulation of guest molecules when chemically modified byperipheral amino acid groups. The cytotoxicity and interaction ofpolypropylenimine dendrimers with DNA as well as the transfectionefficacy of DAB 64 has also been studied.

US Patent Publication No. 20050019923 is based upon the observationthat, contrary to earlier reports, cationic dendrimers, such aspolypropylenimine dendrimers, display suitable properties, such asspecific targeting and low toxicity, for use in the targeted delivery ofbioactive molecules, such as genetic material. In addition, derivativesof the cationic dendrimer also display suitable properties for thetargeted delivery of bioactive molecules. See also, Bioactive Polymers,US published application 20080267903, which discloses “Various polymers,including cationic polyamine polymers and dendrimeric polymers, areshown to possess anti-proliferative activity, and may therefore beuseful for treatment of disorders characterised by undesirable cellularproliferation such as neoplasms and tumours, inflammatory disorders(including autoimmune disorders), psoriasis and atherosclerosis. Thepolymers may be used alone as active agents, or as delivery vehicles forother therapeutic agents, such as drug molecules or nucleic acids forgene therapy. In such cases, the polymers' own intrinsic anti-tumouractivity may complement the activity of the agent to be delivered.” Thedisclosures of these patent publications may be employed in conjunctionwith herein teachings for delivery of nucleic acid-targetingsystem(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of nucleic acid-targetingsystem(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor. Bothsupernegatively and superpositively charged proteins exhibit aremarkable ability to withstand thermally or chemically inducedaggregation. Superpositively charged proteins are also able to penetratemammalian cells. Associating cargo with these proteins, such as plasmidDNA, RNA, or other proteins, can enable the functional delivery of thesemacromolecules into mammalian cells both in vitro and in vivo. DavidLiu's lab reported the creation and characterization of superchargedproteins in 2007 (Lawrence et al., 2007, Journal of the AmericanChemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified+36 GFP protein (or othersuperpositively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116). However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines.

(1) One day before treatment, plate 1×10⁵ cells per well in a 48-wellplate.

(2) On the day of treatment, dilute purified+36 GFP protein in serumfreemedia to a final concentration 200 nM. Add RNA to a final concentrationof 50 nM. Vortex to mix and incubate at room temperature for 10 min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of +36 GFP and RNA, add the protein-RNAcomplexes to cells.

(5) Incubate cells with complexes at 37° C. for 4 h.

(6) Following incubation, aspirate the media and wash three times with20 U/mL heparin PBS. Incubate cells with serum-containing media for afurther 48 h or longer depending upon the assay for activity.

(7) Analyze cells by immunoblot, qPCR, phenotypic assay, or otherappropriate method.

David Liu's lab has further found +36 GFP to be an effective plasmiddelivery reagent in a range of cells. As plasmid DNA is a larger cargothan siRNA, proportionately more +36 GFP protein is required toeffectively complex plasmids. For effective plasmid delivery Applicantshave developed a variant of +36 GFP bearing a C-terminal HA2 peptidetag, a known endosome-disrupting peptide derived from the influenzavirus hemagglutinin protein. The following protocol has been effectivein a variety of cells, but as above it is advised that plasmid DNA andsupercharged protein doses be optimized for specific cell lines anddelivery applications.

(1) One day before treatment, plate 1×10⁵ per well in a 48-well plate.

(2) On the day of treatment, dilute purified

36 GFP protein in serumfree media to a final concentration 2 mM. Add 1mg of plasmid DNA. Vortex to mix and incubate at room temperature for 10min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of

36 GFP and plasmid DNA, gently add the protein-DNA complexes to cells.

(5) Incubate cells with complexes at 37 C for 4 h.

(6) Following incubation, aspirate the media and wash with PBS. Incubatecells in serum-containing media and incubate for a further 24-48 h.

(7) Analyze plasmid delivery (e.g., by plasmid-driven gene expression)as appropriate.

See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA 106,6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752(2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011);Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods ofthe super charged proteins may be used and/or adapted for delivery ofthe nucleic acid-targeting system of the present invention. Thesesystems of Dr. Lui and documents herein in conjunction with hereinteachings can be employed in the delivery of nucleic acid-targetingsystem(s) or component(s) thereof or nucleic acid molecule(s) codingtherefor.

Cell Penetrating Peptides (CPPs)

In yet another embodiment, cell penetrating peptides (CPPs) arecontemplated for the delivery of the CRISPR Cas system. CPPs are shortpeptides that facilitate cellular uptake of various molecular cargo(from nanosize particles to small chemical molecules and large fragmentsof DNA). The term “cargo” as used herein includes but is not limited tothe group consisting of therapeutic agents, diagnostic probes, peptides,nucleic acids, antisense oligonucleotides, plasmids, proteins, particlesincluding nanoparticles, liposomes, chromophores, small molecules andradioactive materials. In aspects of the invention, the cargo may alsocomprise any component of the CRISPR Cas system or the entire functionalCRISPR Cas system. Aspects of the present invention further providemethods for delivering a desired cargo into a subject comprising: (a)preparing a complex comprising the cell penetrating peptide of thepresent invention and a desired cargo, and (b) orally, intraarticularly,intraperitoneally, intrathecally, intrarterially, intranasally,intraparenchymally, subcutaneously, intramuscularly, intravenously,dermally, intrarectally, or topically administering the complex to asubject. The cargo is associated with the peptides either throughchemical linkage via covalent bonds or through non-covalentinteractions.

The function of the CPPs are to deliver the cargo into cells, a processthat commonly occurs through endocytosis with the cargo delivered to theendosomes of living mammalian cells. Cell-penetrating peptides are ofdifferent sizes, amino acid sequences, and charges but all CPPs have onedistinct characteristic, which is the ability to translocate the plasmamembrane and facilitate the delivery of various molecular cargoes to thecytoplasm or an organelle. CPP translocation may be classified intothree main entry mechanisms: direct penetration in the membrane,endocytosis-mediated entry, and translocation through the formation of atransitory structure. CPPs have found numerous applications in medicineas drug delivery agents in the treatment of different diseases includingcancer and virus inhibitors, as well as contrast agents for celllabeling. Examples of the latter include acting as a carrier for GFP,MRI contrast agents, or quantum dots. CPPs hold great potential as invitro and in vivo delivery vectors for use in research and medicine.CPPs typically have an amino acid composition that either contains ahigh relative abundance of positively charged amino acids such as lysineor arginine or has sequences that contain an alternating pattern ofpolar/charged amino acids and non-polar, hydrophobic amino acids. Thesetwo types of structures are referred to as polycationic or amphipathic,respectively. A third class of CPPs are the hydrophobic peptides,containing only apolar residues, with low net charge or have hydrophobicamino acid groups that are crucial for cellular uptake. One of theinitial CPPs discovered was the trans-activating transcriptionalactivator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) which wasfound to be efficiently taken up from the surrounding media by numerouscell types in culture. Since then, the number of known CPPs has expandedconsiderably and small molecule synthetic analogues with more effectiveprotein transduction properties have been generated. CPPs include butare not limited to Penetratin, Tat (48-60), Transportan, and (R-AhX-R4)(Ahx=aminohexanoyl).

U.S. Pat. No. 8,372,951, provides a CPP derived from eosinophil cationicprotein (ECP) which exhibits highly cell-penetrating efficiency and lowtoxicity. Aspects of delivering the CPP with its cargo into a vertebratesubject are also provided. Further aspects of CPPs and their deliveryare described in U.S. Pat. Nos. 8,575,305; 8,614,194 and 8,044,019. CPPscan be used to deliver the CRISPR-Cas system or components thereof. ThatCPPs can be employed to deliver the CRISPR-Cas system or componentsthereof is also provided in the manuscript “Gene disruption bycell-penetrating peptide-mediated delivery of Cas9 protein and guideRNA”, by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, etal. Genome Res. 2014 Apr. 2. [Epub ahead of print], incorporated byreference in its entirety, wherein it is demonstrated that treatmentwith CPP-conjugated recombinant Cas9 protein and CPP-complexed guideRNAs lead to endogenous gene disruptions in human cell lines. In thepaper the Cas9 protein was conjugated to CPP via a thioether bond,whereas the guide RNA was complexed with CPP, forming condensed,positively charged particles. It was shown that simultaneous andsequential treatment of human cells, including embryonic stem cells,dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinomacells, with the modified Cas9 and guide RNA led to efficient genedisruptions with reduced off-target mutations relative to plasmidtransfections.

Implantable Devices

In another embodiment, implantable devices are also contemplated fordelivery of the nucleic acid-targeting system or component(s) thereof ornucleic acid molecule(s) coding therefor. For example, US PatentPublication 20110195123 discloses an implantable medical device whichelutes a drug locally and in prolonged period is provided, includingseveral types of such a device, the treatment modes of implementationand methods of implantation. The device comprising of polymericsubstrate, such as a matrix for example, that is used as the devicebody, and drugs, and in some cases additional scaffolding materials,such as metals or additional polymers, and materials to enhancevisibility and imaging. An implantable delivery device can beadvantageous in providing release locally and over a prolonged period,where drug is released directly to the extracellular matrix (ECM) of thediseased area such as tumor, inflammation, degeneration or forsymptomatic objectives, or to injured smooth muscle cells, or forprevention. One kind of drug is RNA, as disclosed above, and this systemmay be used/and or adapted to the nucleic acid-targeting system of thepresent invention. The modes of implantation in some embodiments areexisting implantation procedures that are developed and used today forother treatments, including brachytherapy and needle biopsy. In suchcases the dimensions of the new implant described in this invention aresimilar to the original implant. Typically, a few devices are implantedduring the same treatment procedure.

US Patent Publication 20110195123, provides a drug delivery implantableor insertable system, including systems applicable to a cavity such asthe abdominal cavity and/or any other type of administration in whichthe drug delivery system is not anchored or attached, comprising abiostable and/or degradable and/or bioabsorbable polymeric substrate,which may for example optionally be a matrix. It should be noted thatthe term “insertion” also includes implantation. The drug deliverysystem is preferably implemented as a “Loder” as described in US PatentPublication 20110195123.

The polymer or plurality of polymers are biocompatible, incorporating anagent and/or plurality of agents, enabling the release of agent at acontrolled rate, wherein the total volume of the polymeric substrate,such as a matrix for example, in some embodiments is optionally andpreferably no greater than a maximum volume that permits a therapeuticlevel of the agent to be reached. As a non-limiting example, such avolume is preferably within the range of 0.1 m³ to 1000 mm³, as requiredby the volume for the agent load. The Loder may optionally be larger,for example when incorporated with a device whose size is determined byfunctionality, for example and without limitation, a knee joint, anintra-uterine or cervical ring and the like.

The drug delivery system (for delivering the composition) is designed insome embodiments to preferably employ degradable polymers, wherein themain release mechanism is bulk erosion; or in some embodiments, nondegradable, or slowly degraded polymers are used, wherein the mainrelease mechanism is diffusion rather than bulk erosion, so that theouter part functions as membrane, and its internal part functions as adrug reservoir, which practically is not affected by the surroundingsfor an extended period (for example from about a week to about a fewmonths). Combinations of different polymers with different releasemechanisms may also optionally be used. The concentration gradient atthe surface is preferably maintained effectively constant during asignificant period of the total drug releasing period, and therefore thediffusion rate is effectively constant (termed “zero mode” diffusion).By the term “constant” it is meant a diffusion rate that is preferablymaintained above the lower threshold of therapeutic effectiveness, butwhich may still optionally feature an initial burst and/or mayfluctuate, for example increasing and decreasing to a certain degree.The diffusion rate is preferably so maintained for a prolonged period,and it can be considered constant to a certain level to optimize thetherapeutically effective period, for example the effective silencingperiod.

The drug delivery system optionally and preferably is designed to shieldthe nucleotide based therapeutic agent from degradation, whetherchemical in nature or due to attack from enzymes and other factors inthe body of the subject.

The drug delivery system of US Patent Publication 20110195123 isoptionally associated with sensing and/or activation appliances that areoperated at and/or after implantation of the device, by non and/orminimally invasive methods of activation and/oracceleration/deceleration, for example optionally including but notlimited to thermal heating and cooling, laser beams, and ultrasonic,including focused ultrasound and/or RF (radiofrequency) methods ordevices.

According to some embodiments of US Patent Publication 20110195123, thesite for local delivery may optionally include target sitescharacterized by high abnormal proliferation of cells, and suppressedapoptosis, including tumors, active and or chronic inflammation andinfection including autoimmune diseases states, degenerating tissueincluding muscle and nervous tissue, chronic pain, degenerative sites,and location of bone fractures and other wound locations for enhancementof regeneration of tissue, and injured cardiac, smooth and striatedmuscle.

The site for implantation of the composition, or target site, preferablyfeatures a radius, area and/or volume that is sufficiently small fortargeted local delivery. For example, the target site optionally has adiameter in a range of from about 0.1 mm to about 5 cm.

The location of the target site is preferably selected for maximumtherapeutic efficacy. For example, the composition of the drug deliverysystem (optionally with a device for implantation as described above) isoptionally and preferably implanted within or in the proximity of atumor environment, or the blood supply associated thereof.

For example, the composition (optionally with the device) is optionallyimplanted within or in the proximity to pancreas, prostate, breast,liver, via the nipple, within the vascular system and so forth.

The target location is optionally selected from the group comprising,consisting essentially of, or consisting of (as non-limiting examplesonly, as optionally any site within the body may be suitable forimplanting a Loder): 1. brain at degenerative sites like in Parkinson orAlzheimer disease at the basal ganglia, white and gray matter; 2. spineas in the case of amyotrophic lateral sclerosis (ALS); 3. uterine cervixto prevent HPV infection; 4. active and chronic inflammatory joints; 5.dermis as in the case of psoriasis; 6. sympathetic and sensoric nervoussites for analgesic effect; 7. Intra osseous implantation; 8. acute andchronic infection sites; 9. Intra vaginal; 10. Inner ear—auditorysystem, labyrinth of the inner ear, vestibular system; 11. Intratracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urinary bladder;14. biliary system; 15. parenchymal tissue including and not limited tothe kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18.dental gums; 19. Intra-articular (into joints); 20. Intra-ocular; 21.Brain tissue; 22. Brain ventricles; 23. Cavities, including abdominalcavity (for example but without limitation, for ovary cancer); 24. Intraesophageal and 25. Intra rectal.

Optionally insertion of the system (for example a device containing thecomposition) is associated with injection of material to the ECM at thetarget site and the vicinity of that site to affect local pH and/ortemperature and/or other biological factors affecting the diffusion ofthe drug and/or drug kinetics in the ECM, of the target site and thevicinity of such a site.

Optionally, according to some embodiments, the release of said agentcould be associated with sensing and/or activation appliances that areoperated prior and/or at and/or after insertion, by non and/or minimallyinvasive and/or else methods of activation and/oracceleration/deceleration, including laser beam, radiation, thermalheating and cooling, and ultrasonic, including focused ultrasound and/orRF (radiofrequency) methods or devices, and chemical activators.

According to other embodiments of US Patent Publication 20110195123, thedrug preferably comprises a RNA, for example for localized cancer casesin breast, pancreas, brain, kidney, bladder, lung, and prostate asdescribed below. Although exemplified with RNAi, many drugs areapplicable to be encapsulated in Loder, and can be used in associationwith this invention, as long as such drugs can be encapsulated with theLoder substrate, such as a matrix for example, and this system may beused and/or adapted to deliver the nucleic acid-targeting system of thepresent invention.

As another example of a specific application, neuro and musculardegenerative diseases develop due to abnormal gene expression. Localdelivery of RNAs may have therapeutic properties for interfering withsuch abnormal gene expression. Local delivery of anti apoptotic, antiinflammatory and anti degenerative drugs including small drugs andmacromolecules may also optionally be therapeutic. In such cases theLoder is applied for prolonged release at constant rate and/or through adedicated device that is implanted separately. All of this may be usedand/or adapted to the nucleic acid-targeting system of the presentinvention.

As yet another example of a specific application, psychiatric andcognitive disorders are treated with gene modifiers. Gene knockdown is atreatment option. Loders locally delivering agents to central nervoussystem sites are therapeutic options for psychiatric and cognitivedisorders including but not limited to psychosis, bi-polar diseases,neurotic disorders and behavioral maladies. The Loders could alsodeliver locally drugs including small drugs and macromolecules uponimplantation at specific brain sites. All of this may be used and/oradapted to the nucleic acid-targeting system of the present invention.

As another example of a specific application, silencing of innate and/oradaptive immune mediators at local sites enables the prevention of organtransplant rejection. Local delivery of RNAs and immunomodulatingreagents with the Loder implanted into the transplanted organ and/or theimplanted site renders local immune suppression by repelling immunecells such as CD8 activated against the transplanted organ. All of thismay be used/and or adapted to the nucleic acid-targeting system of thepresent invention.

As another example of a specific application, vascular growth factorsincluding VEGFs and angiogenin and others are essential forneovascularization. Local delivery of the factors, peptides,peptidomimetics, or suppressing their repressors is an importanttherapeutic modality; silencing the repressors and local delivery of thefactors, peptides, macromolecules and small drugs stimulatingangiogenesis with the Loder is therapeutic for peripheral, systemic andcardiac vascular disease.

The method of insertion, such as implantation, may optionally already beused for other types of tissue implantation and/or for insertions and/orfor sampling tissues, optionally without modifications, or alternativelyoptionally only with non-major modifications in such methods. Suchmethods optionally include but are not limited to brachytherapy methods,biopsy, endoscopy with and/or without ultrasound, such as ERCP,stereotactic methods into the brain tissue, Laparoscopy, includingimplantation with a laparoscope into joints, abdominal organs, thebladder wall and body cavities.

Implantable device technology herein discussed can be employed withherein teachings and hence by this disclosure and the knowledge in theart, CRISPR-Cas system or components thereof or nucleic acid moleculesthereof or encoding or providing components may be delivered via animplantable device.

CRISPR Effector Protein mRNA and Guide RNA

CRISPR effector protein mRNA and guide RNA might also be deliveredseparately. CRISPR effector protein mRNA can be delivered prior to theguide RNA to give time for CRISPR effector protein to be expressed.CRISPR effector protein mRNA might be administered 1-12 hours(preferably around 2-6 hours) prior to the administration of guide RNA.

Alternatively, CRISPR effector protein mRNA and guide RNA can beadministered together. Advantageously, a second booster dose of guideRNA can be administered 1-12 hours (preferably around 2-6 hours) afterthe initial administration of CRISPR effector protein mRNA+guide RNA.

The CRISPR effector protein of the present invention, e.g. aC2c2effector protein is sometimes referred to herein as a CRISPR Enzyme.It will be appreciated that the effector protein is based on or derivedfrom an enzyme, so the term ‘effector protein’ certainly includes‘enzyme’ in some embodiments. However, it will also be appreciated thatthe effector protein may, as required in some embodiments, have DNA orRNA binding, but not necessarily cutting or nicking, activity, includinga dead-Cas effector protein function.

Additional administrations of CRISPR effector protein mRNA and/or guideRNA might be useful to achieve the most efficient levels of genomemodification. In some embodiments, phenotypic alteration is preferablythe result of genome modification when a genetic disease is targeted,especially in methods of therapy and preferably where a repair templateis provided to correct or alter the phenotype.

In some embodiments diseases that may be targeted include thoseconcerned with disease-causing splice defects.

In some embodiments, cellular targets include HemopoieticStem/Progenitor Cells (CD34+); Human T cells; and Eye (retinalcells)—for example photoreceptor precursor cells.

In some embodiments Gene targets include: Human Beta Globin—HBB (fortreating Sickle Cell Anemia, including by stimulating gene-conversion(using closely related HBD gene as an endogenous template)); CD3(T-Cells); and CEP920—retina (eye).

In some embodiments disease targets also include: cancer; Sickle CellAnemia (based on a point mutation); HIV; Beta-Thalassemia; andophthalmic or ocular disease—for example Leber Congenital Amaurosis(LCA)-causing Splice Defect.

In some embodiments delivery methods include: Cationic Lipid Mediated“direct” delivery of Enzyme-Guide complex (RiboNucleoProtein) andelectroporation of plasmid DNA.

Inventive methods can further comprise delivery of templates, such asrepair templates, which may be dsODN or ssODN, see below. Delivery oftemplates may be via the cotemporaneous or separate from delivery of anyor all the CRISPR effector protein or guide and via the same deliverymechanism or different. In some embodiments, it is preferred that thetemplate is delivered together with the guide, and, preferably, also theCRISPR effector protein. An example may be an AAV vector.

Inventive methods can further comprise: (a) delivering to the cell adouble-stranded oligodeoxynucleotide (dsODN) comprising overhangscomplimentary to the overhangs created by said double strand break,wherein said dsODN is integrated into the locus of interest; or —(b)delivering to the cell a single-stranded oligodeoxynucleotide (ssODN),wherein said ssODN acts as a template for homology directed repair ofsaid double strand break. Inventive methods can be for the prevention ortreatment of disease in an individual, optionally wherein said diseaseis caused by a defect in said locus of interest. Inventive methods canbe conducted in vivo in the individual or ex vivo on a cell taken fromthe individual, optionally wherein said cell is returned to theindividual.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of CRISPR effector protein mRNA and guideRNA delivered. Optimal concentrations of CRISPR effector protein mRNAand guide RNA can be determined by testing different concentrations in acellular or animal model and using deep sequencing the analyze theextent of modification at potential off-target genomic loci. Forexample, for the guide sequence targeting 5′-GAGTCCGAGCAGAAGAAGAA-3′(SEQ ID No. 165) in the EMX1 gene of the human genome, deep sequencingcan be used to assess the level of modification at the following twooff-target loci, 1: 5′-GAGTCCTAGCAGGAGAAGAA-3′ (SEQ ID No. 166) and 2:5′-GAGTCTAAGCAGAAGAAGAA-3′ (SEQ ID No. 167). The concentration thatgives the highest level of on-target modification while minimizing thelevel of off-target modification should be chosen for in vivo delivery.

Inducible Systems

In some embodiments, a CRISPR effector protein may form a component ofan inducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR effector protein may be a part of a Light InducibleTranscriptional Effector (LITE) to direct changes in transcriptionalactivity in a sequence-specific manner. The components of a light mayinclude a CRISPR effector protein, a light-responsive cytochromeheterodimer (e.g. from Arabidopsis thaliana), and a transcriptionalactivation/repression domain. Further examples of inducible DNA bindingproteins and methods for their use are provided in U.S. 61/736,465 andU.S. 61/721,283, and WO 2014018423 A2 which is hereby incorporated byreference in its entirety.

Modifying a Target with CRISPR Cas System or Complex (e.g., C2c2-RNAComplex)

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal, and modifying thecell or cells. Culturing may occur at any stage ex vivo. The cell orcells may even be re-introduced into the non-human animal or plant. Forre-introduced cells it is particularly preferred that the cells are stemcells.

In some embodiments, the method comprises allowing a CRISPR complex tobind to the target polynucleotide to effect cleavage of said targetpolynucleotide thereby modifying the target polynucleotide, wherein theCRISPR complex comprises a CRISPR effector protein complexed with aguide sequence hybridized or hybridizable to a target sequence withinsaid target polynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPReffector protein complexed with a guide sequence hybridized orhybridizable to a target sequence within said polynucleotide. Similarconsiderations and conditions apply as above for methods of modifying atarget polynucleotide. In fact, these sampling, culturing andre-introduction options apply across the aspects of the presentinvention.

Indeed, in any aspect of the invention, the CRISPR complex may comprisea CRISPR effector protein complexed with a guide sequence hybridized orhybridizable to a target sequence. Similar considerations and conditionsapply as above for methods of modifying a target polynucleotide.

Thus in any of the non-naturally-occurring CRISPR effector proteinsdescribed herein comprise at least one modification and whereby theeffector protein has certain improved capabilities. In particular, anyof the effector proteins are capable of forming a CRISPR complex with aguide RNA. When such a complex forms, the guide RNA is capable ofbinding to a target polynucleotide sequence and the effector protein iscapable of modifying a target locus. In addition, the effector proteinin the CRISPR complex has reduced capability of modifying one or moreoff-target loci as compared to an unmodified enzyme/effector protein.

In addition, the modified CRISPR enzymes described herein encompassenzymes whereby in the CRISPR complex the effector protein has increasedcapability of modifying the one or more target loci as compared to anunmodified enzyme/effector protein. Such function may be providedseparate to or provided in combination with the above-described functionof reduced capability of modifying one or more off-target loci. Any sucheffector proteins may be provided with any of the further modificationsto the CRISPR effector protein as described herein, such as incombination with any activity provided by one or more associatedheterologous functional domains, any further mutations to reducenuclease activity and the like.

In advantageous embodiments of the invention, the modified CRISPReffector protein is provided with reduced capability of modifying one ormore off-target loci as compared to an unmodified enzyme/effectorprotein and increased capability of modifying the one or more targetloci as compared to an unmodified enzyme/effector protein. Incombination with further modifications to the effector protein,significantly enhanced specificity may be achieved. For example,combination of such advantageous embodiments with one or more additionalmutations is provided wherein the one or more additional mutations arein one or more catalytically active domains. In such effector proteins,enhanced specificity may be achieved due to an improved specificity interms of effector protein activity.

Additional functionalities which may be engineered into modified CRISPReffector proteins as described herein include the following. 1. modifiedCRISPR effector proteins that disrupt RNA:protein interactions withoutaffecting protein tertiary or secondary structure. This includesresidues that contact any part of the RNA:RNA duplex. 2. modified CRISPReffector proteins that weaken intra-protein interactions holding theCRISPR effector in conformation essential for nuclease cutting inresponse to RNA binding (on or off target). For example: a modificationthat mildly inhibits, but still allows, the nuclease conformation of theHNH domain (positioned at the scissile phosphate). 3. modified CRISPReffector proteins that strengthen intra-protein interactions holding theCRISPR effector in a conformation inhibiting nuclease activity inresponse to RNA binding (on or off targets). For example: a modificationthat stabilizes the HNH domain in a conformation away from the scissilephosphate. Any such additional functional enhancement may be provided incombination with any other modification to the CRISPR effector proteinas described in detail elsewhere herein.

Any of the herein described improved functionalities may be made to anyCRISPR effector protein, such as a C2c2 effector protein. However, itwill be appreciated that any of the functionalities described herein maybe engineered into CRISPR effector proteins from other orthologs,including chimeric effector proteins comprising fragments from multipleorthologs.

The invention uses nucleic acids to bind target DNA sequences. This isadvantageous as nucleic acids are much easier and cheaper to producethan proteins, and the specificity can be varied according to the lengthof the stretch where homology is sought. Complex 3-D positioning ofmultiple fingers, for example is not required. The terms“polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid”and “oligonucleotide” are used interchangeably. They refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. Polynucleotides may have anythree dimensional structure, and may perform any function, known orunknown. The following are non-limiting examples of polynucleotides:coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, short interfering RNA (siRNA),short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line. As used herein the term“variant” should be taken to mean the exhibition of qualities that havea pattern that deviates from what occurs in nature. The terms“non-naturally occurring” or “engineered” are used interchangeably andindicate the involvement of the hand of man. The terms, when referringto nucleic acid molecules or polypeptides mean that the nucleic acidmolecule or the polypeptide is at least substantially free from at leastone other component with which they are naturally associated in natureand as found in nature. “Complementarity” refers to the ability of anucleic acid to form hydrogen bond(s) with another nucleic acid sequenceby either traditional Watson-Crick base pairing or other non-traditionaltypes. A percent complementarity indicates the percentage of residues ina nucleic acid molecule which can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence.“Substantially complementary” as used herein refers to a degree ofcomplementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or morenucleotides, or refers to two nucleic acids that hybridize understringent conditions. As used herein, “stringent conditions” forhybridization refer to conditions under which a nucleic acid havingcomplementarity to a target sequence predominantly hybridizes with thetarget sequence, and substantially does not hybridize to non-targetsequences. Stringent conditions are generally sequence-dependent, andvary depending on a number of factors. In general, the longer thesequence, the higher the temperature at which the sequence specificallyhybridizes to its target sequence. Non-limiting examples of stringentconditions are described in detail in Tijssen (1993), LaboratoryTechniques In Biochemistry And Molecular Biology-Hybridization WithNucleic Acid Probes Part I, Second Chapter “Overview of principles ofhybridization and the strategy of nucleic acid probe assay”, Elsevier,N.Y. Where reference is made to a polynucleotide sequence, thencomplementary or partially complementary sequences are also envisaged.These are preferably capable of hybridizing to the reference sequenceunder highly stringent conditions. Generally, in order to maximize thehybridization rate, relatively low-stringency hybridization conditionsare selected: about 20 to 250 C lower than the thermal melting point(T_(m)). The T_(m) is the temperature at which 50% of specific targetsequence hybridizes to a perfectly complementary probe in solution at adefined ionic strength and pH. Generally, in order to require at leastabout 85% nucleotide complementarity of hybridized sequences, highlystringent washing conditions are selected to be about 5 to 150 C lowerthan the T_(m). In order to require at least about 70% nucleotidecomplementarity of hybridized sequences, moderately-stringent washingconditions are selected to be about 15 to 300 C lower than the T_(m).Highly permissive (very low stringency) washing conditions may be as lowas 50° C. below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences. Preferred highly stringent conditions comprise incubation in50% formamide, 5×SSC, and 1% SDS at 420 C, or incubation in 5×SSC and 1%SDS at 650 C, with wash in 0.2×SSC and 0.1% SDS at 65° C.“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions. As used herein, “expressionof a genomic locus” or “gene expression” is the process by whichinformation from a gene is used in the synthesis of a functional geneproduct. The products of gene expression are often proteins, but innon-protein coding genes such as rRNA genes or tRNA genes, the productis functional RNA. The process of gene expression is used by all knownlife—eukaryotes (including multicellular organisms), prokaryotes(bacteria and archaea) and viruses to generate functional products tosurvive. As used herein “expression” of a gene or nucleic acidencompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context. As used herein, “expression” also refers to theprocess by which a polynucleotide is transcribed from a DNA template(such as into and mRNA or other RNA transcript) and/or the process bywhich a transcribed mRNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.” If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell. The terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics. As used herein, the term “domain” or“protein domain” refers to a part of a protein sequence that may existand function independently of the rest of the protein chain. Asdescribed in aspects of the invention, sequence identity is related tosequence homology. Homology comparisons may be conducted by eye, or moreusually, with the aid of readily available sequence comparison programs.These commercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences.

In aspects of the invention the term “guide RNA”, refers to thepolynucleotide sequence comprising one or more of a putative oridentified tracr sequence and a putative or identified crRNA sequence orguide sequence. In particular embodiments, the “guide RNA” comprises aputative or identified crRNA sequence or guide sequence. In furtherembodiments, the guide RNA does not comprise a putative or identifiedtracr sequence.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature. In all aspectsand embodiments, whether they include these terms or not, it will beunderstood that, preferably, the may be optional and thus preferablyincluded or not preferably not included. Furthermore, the terms“non-naturally occurring” and “engineered” may be used interchangeablyand so can therefore be used alone or in combination and one or othermay replace mention of both together. In particular, “engineered” ispreferred in place of “non-naturally occurring” or “non-naturallyoccurring and/or engineered.”

Sequence homologies may be generated by any of a number of computerprograms known in the art, for example BLAST or FASTA, etc. A suitablecomputer program for carrying out such an alignment is the GCG WisconsinBestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984,Nucleic Acids Research 12:387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul etal., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However,it is preferred to use the GCG Bestfit program. Percentage (%) sequencehomology may be calculated over contiguous sequences, i.e., one sequenceis aligned with the other sequence and each amino acid or nucleotide inone sequence is directly compared with the corresponding amino acid ornucleotide in the other sequence, one residue at a time. This is calledan “ungapped” alignment. Typically, such ungapped alignments areperformed only over a relatively short number of residues. Although thisis a very simple and consistent method, it fails to take intoconsideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion may cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without unduly penalizing the overall homology or identityscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximize local homology or identity. However, these more complexmethods assign “gap penalties” to each gap that occurs in the alignmentso that, for the same number of identical amino acids, a sequencealignment with as few gaps as possible ˜reflecting higher relatednessbetween the two compared sequences—may achieve a higher score than onewith many gaps. “Affinity gap costs” are typically used that charge arelatively high cost for the existence of a gap and a smaller penaltyfor each subsequent residue in the gap. This is the most commonly usedgap scoring system. High gap penalties may, of course, produce optimizedalignments with fewer gaps. Most alignment programs allow the gappenalties to be modified. However, it is preferred to use the defaultvalues when using such software for sequence comparisons. For example,when using the GCG Wisconsin Bestfit package the default gap penalty foramino acid sequences is −12 for a gap and −4 for each extension.Calculation of maximum % homology therefore first requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984Nuc. Acids Research 12 p 387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 Short Protocols in Molecular Biology,4th Ed.—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410)and the GENEWORKS suite of comparison tools. Both BLAST and FASTA areavailable for offline and online searching (see Ausubel et al., 1999,Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, forsome applications, it is preferred to use the GCG Bestfit program. A newtool, called BLAST 2 Sequences is also available for comparing proteinand nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50;FEMS Microbiol Lett. 1999 177(1): 187-8 and the website of the NationalCenter for Biotechnology information at the website of the NationalInstitutes for Health). Although the final % homology may be measured interms of identity, the alignment process itself is typically not basedon an all-or-nothing pair comparison. Instead, a scaled similarity scorematrix is generally used that assigns scores to each pair-wisecomparison based on chemical similarity or evolutionary distance. Anexample of such a matrix commonly used is the BLOSUM62 matrix—thedefault matrix for the BLAST suite of programs. GCG Wisconsin programsgenerally use either the public default values or a custom symbolcomparison table, if supplied (see user manual for further details). Forsome applications, it is preferred to use the public default values forthe GCG package, or in the case of other software, the default matrix,such as BLOSUM62. Alternatively, percentage homologies may be calculatedusing the multiple alignment feature in DNASIS™ (Hitachi Software),based on an algorithm, analogous to CLUSTAL (Higgins D G & Sharp P M(1988), Gene 73(1), 237-244). Once the software has produced an optimalalignment, it is possible to calculate % homology, preferably % sequenceidentity. The software typically does this as part of the sequencecomparison and generates a numerical result. The sequences may also havedeletions, insertions or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalentsubstance. Deliberate amino acid substitutions may be made on the basisof similarity in amino acid properties (such as polarity, charge,solubility, hydrophobicity, hydrophilicity, and/or the amphipathicnature of the residues) and it is therefore useful to group amino acidstogether in functional groups. Amino acids may be grouped together basedon the properties of their side chains alone. However, it is more usefulto include mutation data as well. The sets of amino acids thus derivedare likely to be conserved for structural reasons. These sets may bedescribed in the form of a Venn diagram (Livingstone C. D. and Barton G.J. (1993) “Protein sequence alignments: a strategy for the hierarchicalanalysis of residue conservation” Comput. Appl. Biosci. 9: 745-756)(Taylor W. R. (1986) “The classification of amino acid conservation” J.Theor. Biol. 119; 205-218). Conservative substitutions may be made, forexample according to Table 11 below which describes a generally acceptedVenn diagram grouping of amino acids.

TABLE 11 Set Sub-set Hydrophobic F W Y H K M I L V A G C AromaticF W Y H (SEQ ID No. 168) (SEQ ID No. 171) Aliphatic I L V PolarW Y H K R E D C S T N Q Charged H K R E D (SEQ ID No. 169)(SEQ ID No. 172) Positively charged H K R Negatively charged E D SmallV C A G S P T N D Tiny A G S (SEQ ID No. 170)

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Embodiments of the invention include sequences (both polynucleotide orpolypeptide) which may comprise homologous substitution (substitutionand replacement are both used herein to mean the interchange of anexisting amino acid residue or nucleotide, with an alternative residueor nucleotide) that may occur i.e., like-for-like substitution in thecase of amino acids such as basic for basic, acidic for acidic, polarfor polar, etc. Non-homologous substitution may also occur i.e., fromone class of residue to another or alternatively involving the inclusionof unnatural amino acids such as ornithine (hereinafter referred to asZ), diaminobutyric acid ornithine (hereinafter referred to as B),norleucine ornithine (hereinafter referred to as O), pyriylalanine,thienylalanine, naphthylalanine and phenylglycine. Variant amino acidsequences may include suitable spacer groups that may be insertedbetween any two amino acid residues of the sequence including alkylgroups such as methyl, ethyl or propyl groups in addition to amino acidspacers such as glycine or β-alanine residues. A further form ofvariation, which involves the presence of one or more amino acidresidues in peptoid form, may be well understood by those skilled in theart. For the avoidance of doubt, “the peptoid form” is used to refer tovariant amino acid residues wherein the α-carbon substituent group is onthe residue's nitrogen atom rather than the α-carbon. Processes forpreparing peptides in the peptoid form are known in the art, for exampleSimon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, TrendsBiotechnol. (1995) 13(4), 132-134.

Homology modelling: Corresponding residues in other CRISPR orthologs canbe identified by the methods of Zhang et al., 2012 (Nature; 490(7421):556-60) and Chen et al., 2015 (PLoS Comput Biol; 11(5): e1004248)—acomputational protein-protein interaction (PPI) method to predictinteractions mediated by domain-motif interfaces. PrePPI (PredictingPPI), a structure based PPI prediction method, combines structuralevidence with non-structural evidence using a Bayesian statisticalframework. The method involves taking a pair a query proteins and usingstructural alignment to identify structural representatives thatcorrespond to either their experimentally determined structures orhomology models. Structural alignment is further used to identify bothclose and remote structural neighbors by considering global and localgeometric relationships. Whenever two neighbors of the structuralrepresentatives form a complex reported in the Protein Data Bank, thisdefines a template for modelling the interaction between the two queryproteins. Models of the complex are created by superimposing therepresentative structures on their corresponding structural neighbor inthe template. This approach is further described in Dey et al., 2013(Prot Sci; 22: 359-66).

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR.

In certain aspects the invention involves vectors. A used herein, a“vector” is a tool that allows or facilitates the transfer of an entityfrom one environment to another. It is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. Generally, avector is capable of replication when associated with the proper controlelements. In general, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. Vectors include, but are not limited to, nucleic acidmolecules that are single-stranded, double-stranded, or partiallydouble-stranded; nucleic acid molecules that comprise one or more freeends, no free ends (e.g., circular); nucleic acid molecules thatcomprise DNA, RNA, or both; and other varieties of polynucleotides knownin the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses(AAVs)). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g., bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

Aspects of the invention relate to bicistronic vectors for guide RNA andwild type, modified or mutated CRISPR effector proteins/enzymes (e.g.C2c2). Bicistronic expression vectors guide RNA and wild type, modifiedor mutated CRISPR effector proteins/enzymes (e.g. C2c2) are preferred.In general and particularly in this embodiment and wild type, modifiedor mutated CRISPR effector proteins/enzymes (e.g. C2c2) is preferablydriven by the CBh promoter. The RNA may preferably be driven by a PolIII promoter, such as a U6 promoter. Ideally the two are combined.

In some embodiments, a loop in the guide RNA is provided. This may be astem loop or a tetra loop. The loop is preferably GAAA, but it is notlimited to this sequence or indeed to being only 4 bp in length. Indeed,preferred loop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG.

In practicing any of the methods disclosed herein, a suitable vector canbe introduced to a cell or an embryo via one or more methods known inthe art, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).With regards to regulatory sequences, mention is made of U.S. patentapplication Ser. No. 10/491,026, the contents of which are incorporatedby reference herein in their entirety. With regards to promoters,mention is made of PCT publication WO 2011/028929 and U.S. applicationSer. No. 12/511,940, the contents of which are incorporated by referenceherein in their entirety.

Vectors can be designed for expression of CRISPR transcripts (e.g.,nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g., amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

In some embodiments, a vector is a yeast expression vector. Examples ofvectors for expression in yeast Saccharomyces cerivisae include pYepSec1(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan andHerskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (InVitrogen Corp, San Diego, Calif.).

In some embodiments, a vector drives protein expression in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546). With regards to theseprokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No.6,750,059, the contents of which are incorporated by reference herein intheir entirety. Other embodiments of the invention may relate to the useof viral vectors, with regards to which mention is made of U.S. patentapplication Ser. No. 13/092,085, the contents of which are incorporatedby reference herein in their entirety. Tissue-specific regulatoryelements are known in the art and in this regard, mention is made ofU.S. Pat. No. 7,776,321, the contents of which are incorporated byreference herein in their entirety.

In some embodiments, a regulatory element is operably linked to one ormore elements of a CRISPR system so as to drive expression of the one ormore elements of the CRISPR system. In general, CRISPRs (ClusteredRegularly Interspaced Short Palindromic Repeats), also known as SPIDRs(SPacer Interspersed Direct Repeats), constitute a family of DNA locithat are usually specific to a particular bacterial species. The CRISPRlocus comprises a distinct class of interspersed short sequence repeats(SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol.,169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556[1989]), and associated genes. Similar interspersed SSRs have beenidentified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena,and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol.,10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999];Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica etal., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differfrom other SSRs by the structure of the repeats, which have been termedshort regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ.Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246[2000]). In general, the repeats are short elements that occur inclusters that are regularly spaced by unique intervening sequences witha substantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In general, “nucleic acid-targeting system” as used in the presentapplication refers collectively to transcripts and other elementsinvolved in the expression of or directing the activity of nucleicacid-targeting CRISPR-associated (“Cas”) genes (also referred to hereinas an effector protein), including sequences encoding a nucleicacid-targeting Cas (effector) protein and a guide RNA (comprising crRNAsequence and a trans-activating CRISPR/Cas system RNA (tracrRNA)sequence), or other sequences and transcripts from a nucleicacid-targeting CRISPR locus. In some embodiments, one or more elementsof a nucleic acid-targeting system are derived from a Type V/Type VInucleic acid-targeting CRISPR system. In some embodiments, one or moreelements of a nucleic acid-targeting system is derived from a particularorganism comprising an endogenous nucleic acid-targeting CRISPR system.In general, a nucleic acid-targeting system is characterized by elementsthat promote the formation of a nucleic acid-targeting complex at thesite of a target sequence. In the context of formation of a nucleicacid-targeting complex, “target sequence” refers to a sequence to whicha guide sequence is designed to have complementarity, wherehybridization between a target sequence and a guide RNA promotes theformation of a DNA or RNA-targeting complex. Full complementarity is notnecessarily required, provided there is sufficient complementarity tocause hybridization and promote formation of a nucleic acid-targetingcomplex. A target sequence may comprise RNA polynucleotides. In someembodiments, a target sequence is located in the nucleus or cytoplasm ofa cell. In some embodiments, the target sequence may be within anorganelle of a eukaryotic cell, for example, mitochondrion orchloroplast. A sequence or template that may be used for recombinationinto the targeted locus comprising the target sequences is referred toas an “editing template” or “editing RNA” or “editing sequence”. Inaspects of the invention, an exogenous template RNA may be referred toas an editing template. In an aspect of the invention the recombinationis homologous recombination.

Typically, in the context of an endogenous nucleic acid-targetingsystem, formation of a nucleic acid-targeting complex (comprising aguide RNA hybridized to a target sequence and complexed with one or morenucleic acid-targeting effector proteins) results in cleavage of one orboth RNA strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, or more base pairs from) the target sequence. In someembodiments, one or more vectors driving expression of one or moreelements of a nucleic acid-targeting system are introduced into a hostcell such that expression of the elements of the nucleic acid-targetingsystem direct formation of a nucleic acid-targeting complex at one ormore target sites. For example, a nucleic acid-targeting effectorprotein and a guide RNA could each be operably linked to separateregulatory elements on separate vectors. Alternatively, two or more ofthe elements expressed from the same or different regulatory elements,may be combined in a single vector, with one or more additional vectorsproviding any components of the nucleic acid-targeting system notincluded in the first vector. nucleic acid-targeting system elementsthat are combined in a single vector may be arranged in any suitableorientation, such as one element located 5′ with respect to (“upstream”of) or 3′ with respect to (“downstream” of) a second element. The codingsequence of one element may be located on the same or opposite strand ofthe coding sequence of a second element, and oriented in the same oropposite direction. In some embodiments, a single promoter drivesexpression of a transcript encoding a nucleic acid-targeting effectorprotein and a guide RNA embedded within one or more intron sequences(e.g. each in a different intron, two or more in at least one intron, orall in a single intron). In some embodiments, the nucleic acid-targetingeffector protein and guide RNA are operably linked to and expressed fromthe same promoter.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a nucleic acid-targeting complex to the target sequence. In someembodiments, the degree of complementarity between a guide sequence andits corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, SanDiego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq(available at maq.sourceforge.net). In some embodiments, a guidesequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75,or more nucleotides in length. In some embodiments, a guide sequence isless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length. The ability of a guide sequence to directsequence-specific binding of a nucleic acid-targeting complex to atarget sequence may be assessed by any suitable assay. For example, thecomponents of a nucleic acid-targeting system sufficient to form anucleic acid-targeting complex, including the guide sequence to betested, may be provided to a host cell having the corresponding targetsequence, such as by transfection with vectors encoding the componentsof the nucleic acid-targeting CRISPR sequence, followed by an assessmentof preferential cleavage within or in the vicinity of the targetsequence, such as by Surveyor assay as described herein. Similarly,cleavage of a target polynucleotide sequence (or a sequence in thevicinity thereof) may be evaluated in a test tube by providing thetarget sequence, components of a nucleic acid-targeting complex,including the guide sequence to be tested and a control guide sequencedifferent from the test guide sequence, and comparing binding or rate ofcleavage at or in the vicinity of the target sequence between the testand control guide sequence reactions. Other assays are possible, andwill occur to those skilled in the art.

A guide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a gene transcriptor mRNA.

In some embodiments, the target sequence is a sequence within a genomeof a cell.

In some embodiments, a guide sequence is selected to reduce the degreeof secondary structure within the guide sequence. Secondary structuremay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology27(12): 1151-62). Further algorithms may be found in U.S. applicationSer. No. TBA (attorney docket 44790.11.2022; Broad ReferenceBI-2013/004A); incorporated herein by reference.

In some embodiments, the nucleic acid-targeting effector protein is partof a fusion protein comprising one or more heterologous protein domains(e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moredomains in addition to the nucleic acid-targeting effector protein). Insome embodiments, the CRISPR effector protein/enzyme is part of a fusionprotein comprising one or more heterologous protein domains (e.g. aboutor more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains inaddition to the CRISPR enzyme). A CRISPR effector protein/enzyme fusionprotein may comprise any additional protein sequence, and optionally alinker sequence between any two domains. Examples of protein domainsthat may be fused to an effector protein include, without limitation,epitope tags, reporter gene sequences, and protein domains having one ormore of the following activities: methylase activity, demethylaseactivity, transcription activation activity, transcription repressionactivity, transcription release factor activity, histone modificationactivity, RNA cleavage activity and nucleic acid binding activity.Non-limiting examples of epitope tags include histidine (His) tags, V5tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-Gtags, and thioredoxin (Trx) tags. Examples of reporter genes include,but are not limited to, glutathione-S-transferase (GST), horseradishperoxidase (HRP), chloramphenicol acetyltransferase (CAT)beta-galactosidase, beta-glucuronidase, luciferase, green fluorescentprotein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellowfluorescent protein (YFP), and autofluorescent proteins including bluefluorescent protein (BFP). A nucleic acid-targeting effector protein maybe fused to a gene sequence encoding a protein or a fragment of aprotein that bind DNA molecules or bind other cellular molecules,including but not limited to maltose binding protein (MBP), S-tag, Lex ADNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, andherpes simplex virus (HSV) BP16 protein fusions. Additional domains thatmay form part of a fusion protein comprising a nucleic acid-targetingeffector protein are described in US20110059502, incorporated herein byreference. In some embodiments, a tagged nucleic acid-targeting effectorprotein is used to identify the location of a target sequence.

In some embodiments, a CRISPR enzyme may form a component of aninducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR enzyme may be a part of a Light Inducible TranscriptionalEffector (LITE) to direct changes in transcriptional activity in asequence-specific manner. The components of a light may include a CRISPRenzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283 and WO 2014/018423 andU.S. Pat. Nos. 8,889,418, 8,895,308, US20140186919, US20140242700,US20140273234, US20140335620, WO2014093635, which is hereby incorporatedby reference in its entirety.

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and organisms (such asanimals, plants, or fungi) comprising or produced from such cells. Insome embodiments, a nucleic acid-targeting effector protein incombination with (and optionally complexed with) a guide RNA isdelivered to a cell. Conventional viral and non-viral based genetransfer methods can be used to introduce nucleic acids in mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding components of a nucleic acid-targeting system to cells inculture, or in a host organism. Non-viral vector delivery systemsinclude DNA plasmids, RNA (e.g. a transcript of a vector describedherein), naked nucleic acid, and nucleic acid complexed with a deliveryvehicle, such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology, Doerfler and Bohm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids takes advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700). In applications where transient expression ispreferred, adenoviral based systems may be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors may also be used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Functional Alteration

The use of the CRISPR system of the present invention to preciselydeliver functional domains, to activate or repress genes or to alterepigenetic state by precisely altering the methylation site on a aspecific locus of interest, can be with one or more guide RNAs appliedto a single cell or population of cells or with a library applied togenome in a pool of cells ex vivo or in vivo comprising theadministration or expression of a library comprising a plurality ofguide RNAs (sgRNAs) and wherein wherein the CRISPR complex comprisingthe CRISPR effector protein is modified to comprise a heterologousfunctional domain. In an aspect the invention provides a method forscreening a genome/transcriptome comprising the administration to a hostor expression in a host in vivo of a library. In an aspect the inventionprovides a method as herein discussed further comprising an activatoradministered to the host or expressed in the host. In an aspect theinvention provides a method as herein discussed wherein the activator isattached to a CRISPR effector protein. In an aspect the inventionprovides a method as herein discussed wherein the activator is attachedto the N terminus or the C terminus of the CRISPR effector protein. Inan aspect the invention provides a method as herein discussed whereinthe activator is attached to a sgRNA loop. In an aspect the inventionprovides a method as herein discussed further comprising a repressoradministered to the host or expressed in the host. In an aspect theinvention provides a method as herein discussed, wherein the screeningcomprises affecting and detecting gene activation, gene inhibition, orcleavage in the locus.

In an aspect, the invention provides efficient on-target activity andminimizes off target activity. In an aspect, the invention providesefficient on-target cleavage by CRISPR effector protein and minimizesoff-target cleavage by the CRISPR effector protein. In an aspect, theinvention provides guide specific binding of CRISPR effector protein ata gene locus without DNA cleavage. Accordingly, in an aspect, theinvention provides target-specific gene regulation. In an aspect, theinvention provides guide specific binding of CRISPR effector protein ata gene locus without DNA cleavage. Accordingly, in an aspect, theinvention provides for cleavage at one locus and gene regulation at adifferent locus using a single CRISPR effector protein. In an aspect,the invention provides orthogonal activation and/or inhibition and/orcleavage of multiple targets using one or more CRISPR effector proteinand/or enzyme.

In an aspect the invention provides a method as herein discussed,wherein the host is a eukaryotic cell. In an aspect the inventionprovides a method as herein discussed, wherein the host is a mammaliancell. In an aspect the invention provides a method as herein discussed,wherein the host is a non-human eukaryote. In an aspect the inventionprovides a method as herein discussed, wherein the non-human eukaryoteis a non-human mammal. In an aspect the invention provides a method asherein discussed, wherein the non-human mammal is a mouse. An aspect theinvention provides a method as herein discussed comprising the deliveryof the CRISPR effector protein complexes or component(s) thereof ornucleic acid molecule(s) coding therefor, wherein said nucleic acidmolecule(s) are operatively linked to regulatory sequence(s) andexpressed in vivo. In an aspect the invention provides a method asherein discussed wherein the expressing in vivo is via a lentivirus, anadenovirus, or an AAV. In an aspect the invention provides a method asherein discussed wherein the delivery is via a particle, a nanoparticle,a lipid or a cell penetrating peptide (CPP).

In an aspect the invention provides a pair of CRISPR complexescomprising CRISPR effector protein, each comprising a guide RNA (sgRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, wherein at least one loop ofeach sgRNA is modified by the insertion of distinct RNA sequence(s) thatbind to one or more adaptor proteins, and wherein the adaptor protein isassociated with one or more functional domains, wherein each sgRNA ofeach CRISPR effector protein complex comprises a functional domainhaving a DNA cleavage activity.

In an aspect the invention provides a method for cutting a targetsequence in a locus of interest comprising delivery to a cell of theCRISPR effector protein complexes or component(s) thereof or nucleicacid molecule(s) coding therefor, wherein said nucleic acid molecule(s)are operatively linked to regulatory sequence(s) and expressed in vivo.In an aspect the invention provides a method as herein-discussed whereinthe delivery is via a lentivirus, an adenovirus, or an AAV.

In an aspect the invention provides a library, method or complex asherein-discussed wherein the sgRNA is modified to have at least onenon-coding functional loop, e.g., wherein the at least one non-codingfunctional loop is repressive; for instance, wherein the at least onenon-coding functional loop comprises Alu.

In one aspect, the invention provides a method for altering or modifyingexpression of a gene product. The said method may comprise introducinginto a cell containing and expressing a DNA molecule encoding the geneproduct an engineered, non-naturally occurring CRISPR system comprisinga CRISPR effector protein and guide RNA that targets the RNA molecule,whereby the guide RNA targets the RNA target molecule encoding the geneproduct and the CRISPR effector protein cleaves the RNA moleculeencoding the gene product, whereby expression of the gene product isaltered; and, wherein the CRISPR effector protein and the guide RNA donot naturally occur together. The invention comprehends the guide RNAcomprising a guide sequence linked to a direct repeat sequence. Theinvention further comprehends the CRISPR effector protein being codonoptimized for expression in a Eukaryotic cell. In a preferred embodimentthe Eukaryotic cell is a mammalian cell and in a more preferredembodiment the mammalian cell is a human cell. In a further embodimentof the invention, the expression of the gene product is decreased.

In some embodiments, one or more functional domains are associated withthe CRISPR effector protein. In some embodiments, one or more functionaldomains are associated with an adaptor protein, for example as used withthe modified guides of Konnerman et al. (Nature 517, 583-588, 29 Jan.2015). In some embodiments, one or more functional domains areassociated with a dead sgRNA (dRNA). In some embodiments, a dRNA complexwith active CRISPR effector protein directs gene regulation by afunctional domain at on gene locus while an sgRNA directs DNA cleavageby the active CRISPR effector protein at another locus, for example asdescribed analogously in CRISPR-Cas9 systems by Dahlman et al.,‘Orthogonal gene control with a catalytically active Cas9 nuclease’ (inpress). In some embodiments, dRNAs are selected to maximize selectivityof regulation for a gene locus of interest compared to off-targetregulation. In some embodiments, dRNAs are selected to maximize targetgene regulation and minimize target cleavage

In certain embodiments, the (heterologous) functional domain is atranslational repressor.

For the purposes of the following discussion, reference to a functionaldomain could be a functional domain associated with the CRISPR effectorprotein or a functional domain associated with the adaptor protein.

In some embodiments, the one or more functional domains is an NLS(Nuclear Localization Sequence) or an NES (Nuclear Export Signal). Insome embodiments, the one or more functional domains is atranscriptional activation domain comprises VP64, p65, MyoD1, HSF1, RTA,SET7/9 and a histone acetyltransferase. Other references herein toactivation (or activator) domains in respect of those associated withthe CRISPR enzyme include any known transcriptional activation domainand specifically VP64, p65, MyoD1, HSF1, RTA, SET7/9 or a histoneacetyltransferase.

In some embodiments, the one or more functional domains is atranscriptional repressor domain. In some embodiments, thetranscriptional repressor domain is a KRAB domain. In some embodiments,the transcriptional repressor domain is a NuE domain, NcoR domain, SIDdomain or a SID4X domain.

In some embodiments, the one or more functional domains have one or moreactivities comprising translation activation activity, translationrepression activity, methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,RNA cleavage activity, DNA cleavage activity, DNA integration activityor nucleic acid binding activity.

In some embodiments, the DNA cleavage activity is due to a nuclease. Insome embodiments, the nuclease comprises a Fok1 nuclease. See, “DimericCRISPR RNA-guided FokI nucleases for highly specific genome editing”,Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. Foden,Vishal Thapar, Deepak Reyon, Mathew J. Goodwin, Martin J. Aryee, J.Keith Joung Nature Biotechnology 32(6): 569-77 (2014), relates todimeric RNA-guided FokI Nucleases that recognize extended sequences andcan edit endogenous genes with high efficiencies in human cells.

In some embodiments, the one or more functional domains is attached tothe CRISPR effector protein so that upon binding to the sgRNA and targetthe functional domain is in a spatial orientation allowing for thefunctional domain to function in its attributed function.

In some embodiments, the one or more functional domains is attached tothe adaptor protein so that upon binding of the CRISPR effector proteinto the sgRNA and target, the functional domain is in a spatialorientation allowing for the functional domain to function in itsattributed function.

In an aspect the invention provides a composition as herein discussedwherein the one or more functional domains is attached to the CRISPReffector protein or adaptor protein via a linker, optionally a GlySerlinker, as discussed herein.

It is also preferred to target endogenous (regulatory) control elements,such as involved in translation, stability, etc. Targeting of knowncontrol elements can be used to activate or repress the gene ofinterest. Targeting of putative control elements on the other hand canbe used as a means to verify such elements (by measuring the translationof the gene of interest) or to detect novel control elements (Inaddition, targeting of putative control elements can be useful in thecontext of understanding genetic causes of disease. Many mutations andcommon SNP variants associated with disease phenotypes are locatedoutside coding regions. Targeting of such regions with either theactivation or repression systems described herein can be followed byreadout of transcription of either a) a set of putative targets (e.g. aset of genes located in closest proximity to the control element) or b)whole-transcriptome readout by e.g. RNAseq or microarray. This wouldallow for the identification of likely candidate genes involved in thedisease phenotype. Such candidate genes could be useful as novel drugtargets.

Histone acetyltransferase (HAT) inhibitors are mentioned herein.However, an alternative in some embodiments is for the one or morefunctional domains to comprise an acetyltransferase, preferably ahistone acetyltransferase. These are useful in the field of epigenomics,for example in methods of interrogating the epigenome. Methods ofinterrogating the epigenome may include, for example, targetingepigenomic sequences. Targeting epigenomic sequences may include theguide being directed to an epigenomic target sequence. Epigenomic targetsequence may include, in some embodiments, include a promoter, silenceror an enhancer sequence.

Use of a functional domain linked to a CRISPR effector protein asdescribed herein, preferably a dead-CRISPR effector protein, morepreferably a dead-FnCRISPR effector protein, to target epigenomicsequences can be used to activate or repress promoters, silencer orenhancers.

Examples of acetyltransferases are known but may include, in someembodiments, histone acetyltransferases. In some embodiments, thehistone acetyltransferase may comprise the catalytic core of the humanacetyltransferase p300 (Gerbasch & Reddy, Nature Biotech 6 Apr. 2015).

In some preferred embodiments, the functional domain is linked to adead-CRISPR effector protein to target and activate epigenomic sequencessuch as promoters or enhancers. One or more guides directed to suchpromoters or enhancers may also be provided to direct the binding of theCRISPR enzyme to such promoters or enhancers.

In certain embodiments, the RNA targeting effector protein of theinvention can be used to interfere with co-transcriptional modificationsof DNA/chromatin structure, RNA-directed DNA methylation, orRNA-directed silencing/activation of DNA/chromatin. RNA-directed DNAmethylation (RdDM) is an epigenetic process first discovered in plants.During RdDM, double-stranded RNAs (dsRNAs) are processed to 21-24nucleotide small interfering RNAs (siRNAs) and guide methylation ofhomologous DNA loci. Besides RNA molecules, a plethora of proteins areinvolved in the establishment of RdDM, like Argonautes, DNAmethyltransferases, chromatin remodelling complexes and theplant-specific PolIV and PolV. All these act in concert to add amethyl-group at the 5′ position of cytosines. Small RNAs can modify thechromatin structure and silence transcription by guidingArgonaute-containing complexes to complementary nascent (non-coding) RNAtrancripts. Subsequently the recruitment of chromatin-modifyingcomplexes, including histone and DNA methyltransferases, is mediated.The RNA targeting effector protein of the invention may be used totarget such small RNAs and interfere in interactions between these smallRNAs and the nascent non-coding transcripts.

The term “associated with” is used here in relation to the associationof the functional domain to the CRISPR effector protein or the adaptorprotein. It is used in respect of how one molecule ‘associates’ withrespect to another, for example between an adaptor protein and afunctional domain, or between the CRISPR effector protein and afunctional domain. In the case of such protein-protein interactions,this association may be viewed in terms of recognition in the way anantibody recognizes an epitope. Alternatively, one protein may beassociated with another protein via a fusion of the two, for instanceone subunit being fused to another subunit. Fusion typically occurs byaddition of the amino acid sequence of one to that of the other, forinstance via splicing together of the nucleotide sequences that encodeeach protein or subunit. Alternatively, this may essentially be viewedas binding between two molecules or direct linkage, such as a fusionprotein. In any event, the fusion protein may include a linker betweenthe two subunits of interest (i.e. between the enzyme and the functionaldomain or between the adaptor protein and the functional domain). Thus,in some embodiments, the CRISPR effector protein or adaptor protein isassociated with a functional domain by binding thereto. In otherembodiments, the CRISPR effector protein or adaptor protein isassociated with a functional domain because the two are fused together,optionally via an intermediate linker.

CRISPR Effector Protein Complexes can be Used in Plants

The CRISPR effector protein system(s) (e.g., single or multiplexed) canbe used in conjunction with recent advances in crop genomics. Thesystems described herein can be used to perform efficient and costeffective plant gene or genome interrogation or editing ormanipulation—for instance, for rapid investigation and/or selectionand/or interrogations and/or comparison and/or manipulations and/ortransformation of plant genes or genomes; e.g., to create, identify,develop, optimize, or confer trait(s) or characteristic(s) to plant(s)or to transform a plant genome. There can accordingly be improvedproduction of plants, new plants with new combinations of traits orcharacteristics or new plants with enhanced traits. The CRISPR effectorprotein system(s) can be used with regard to plants in Site-DirectedIntegration (SDI) or Gene Editing (GE) or any Near Reverse Breeding(NRB) or Reverse Breeding (RB) techniques. Aspects of utilizing theherein described CRISPR effector protein systems may be analogous to theuse of the CRISPR-Cas (e.g. CRISPR-Cas9) system in plants, and mentionis made of the University of Arizona web site “CRISPR-PLANT”(http://www.genome.arizona.edu/crispr/) (supported by Penn State andAGI). Embodiments of the invention can be used in genome editing inplants or where RNAi or similar genome editing techniques have been usedpreviously; see, e.g., Nekrasov, “Plant genome editing made easy:targeted mutagenesis in model and crop plants using the CRISPR-Cassystem,” Plant Methods 2013, 9:39 (doi:10.1186/1746-4811-9-39); Brooks,“Efficient gene editing in tomato in the first generation using theCRISPR-Cas9 system,” Plant Physiology September 2014 pp 114.247577;Shan, “Targeted genome modification of crop plants using a CRISPR-Cassystem,” Nature Biotechnology 31, 686-688 (2013); Feng, “Efficientgenome editing in plants using a CRISPR/Cas system,” Cell Research(2013) 23:1229-1232. doi:10.1038/cr.2013.114; published online 20 Aug.2013; Xie, “RNA-guided genome editing in plants using a CRISPR-Cassystem,” Mol Plant. 2013 November; 6(6):1975-83. doi: 10.1093/mp/sst119.Epub 2013 Aug. 17; Xu, “Gene targeting using the Agrobacteriumtumefaciens-mediated CRISPR-Cas system in rice,” Rice 2014, 7:5 (2014),Zhou et al., “Exploiting SNPs for biallelic CRISPR mutations in theoutcrossing woody perennial Populus reveals 4-coumarate: CoA ligasespecificity and Redundancy,” New Phytologist (2015) (Forum) 1-4(available online only at www.newphytologist.com); Caliando et al,“Targeted DNA degradation using a CRISPR device stably carried in thehost genome, NATURE COMMUNICATIONS 6:6989, DOI: 10.1038/ncomms7989,www.nature.com/naturecommunications DOI: 10.1038/ncomms7989; U.S. Pat.No. 6,603,061—Agrobacterium-Mediated Plant Transformation Method; U.S.Pat. No. 7,868,149—Plant Genome Sequences and Uses Thereof and US2009/0100536—Transgenic Plants with Enhanced Agronomic Traits, all thecontents and disclosure of each of which are herein incorporated byreference in their entirety. In the practice of the invention, thecontents and disclosure of Morrell et al “Crop genomics: advances andapplications,” Nat Rev Genet. 2011 Dec. 29; 13(2):85-96; each of whichis incorporated by reference herein including as to how hereinembodiments may be used as to plants. Accordingly, reference herein toanimal cells may also apply, mutatis mutandis, to plant cells unlessotherwise apparent; and, the enzymes herein having reduced off-targeteffects and systems employing such enzymes can be used in plantapplciations, including those mentioned herein.

In an aspect, the RNA targeting effector protein of the invention can beused for antiviral activity in plants, in particular against RNAviruses. The effector protein can be targeted to the viral RNA using asuitable guide RNA selective for a selected viral RNA sequence. Inparticular, the effector protein may be an active nuclease that cleavesRNA, such as single stranded RNA. provided is therefore the use of anRNA targeting effector protein of the invention as an antiviral agent.Examples of viruses that can be counteracted in this way include, butare not limited to, Tobacco mosaic virus (TMV), Tomato spotted wiltvirus (TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY),Cauliflower mosaic virus (CaMV) (RT virus), Plum pox virus (PPV), Bromemosaic virus (BMV) and Potato virus X (PVX).

Sugano et al. (Plant Cell Physiol. 2014 March; 55(3):475-81. doi:10.1093/pcp/pcu014. Epub 2014 Jan. 18) reports the application ofCRISPR-Cas9 to targeted mutagenesis in the liverwort Marchantiapolymorpha L., which has emerged as a model species for studying landplant evolution. The U6 promoter of M. polymorpha was identified andcloned to express the gRNA. The target sequence of the gRNA was designedto disrupt the gene encoding auxin response factor 1 (ARF1) in M.polymorpha. Using Agrobacterium-mediated transformation, Sugano et al.isolated stable mutants in the gametophyte generation of M. polymorpha.CRISPR-Cas9-based site-directed mutagenesis in vivo was achieved usingeither the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoterto express Cas9. Isolated mutant individuals showing an auxin-resistantphenotype were not chimeric. Moreover, stable mutants were produced byasexual reproduction of T1 plants. Multiple arf1 alleles were easilyestablished using CRIPSR/Cas9-based targeted mutagenesis. The CRISPRsystems of the present invention can be used to regulate the same aswell as other genes, and like expression control systesm such as RNAiand siRNA, the method of the invention can be inducible and reversible.

Kabadi et al. (Nucleic Acids Res. 2014 Oct. 29; 42(19):e147. doi:10.1093/nar/gku749. Epub 2014 Aug. 13) developed a single lentiviralsystem to express a Cas9 variant, a reporter gene and up to four sgRNAsfrom independent RNA polymerase III promoters that are incorporated intothe vector by a convenient Golden Gate cloning method. Each sgRNA wasefficiently expressed and can mediate multiplex gene editing andsustained transcriptional activation in immortalized and primary humancells. The instant invention can be used to regulate the plant genes ofKabadi.

Xing et al. (BMC Plant Biology 2014, 14:327) developed a CRISPR-Cas9binary vector set based on the pGreen or pCAMBIA backbone, as well as agRNA. This toolkit requires no restriction enzymes besides BsaI togenerate final constructs harboring maize-codon optimized Cas9 and oneor more gRNAs with high efficiency in as little as one cloning step. Thetoolkit was validated using maize protoplasts, transgenic maize lines,and transgenic Arabidopsis lines and was shown to exhibit highefficiency and specificity. More importantly, using this toolkit,targeted mutations of three Arabidopsis genes were detected intransgenic seedlings of the T1 generation. Moreover, the multiple-genemutations could be inherited by the next generation. (guide RNA) modulevector set, as a toolkit for multiplex genome editing in plants. TheCRISPR systems and proteins of the instant invention may be used totarget the genes targeted by Xing.

The CRISPR systems of the invention may be used in the treatment,prevention, suppression, and/or alleviation of of plant viruspathogenesis, replication, proparation, viremia, viral load or titerand/or infection. Gambino et al. (Phytopathology. 2006 November;96(11):1223-9. doi: 10.1094/PHYTO-96-1223) relied on amplification andmultiplex PCR for simultaneous detection of nine grapevine viruses. TheCRISPR systems and proteins of the instant invention may similarly beused to detect multiple targets in a host. Moreover, the systems of theinvention can be used to simultaneously knock down viral gene expressionin valuable cultivars, and prevent activation or further infection bytargeting expressed vial RNA.

Murray et al. (Proc Biol Sci. 2013 Jun. 26; 280(1765):20130965. doi:10.1098/rspb.2013.0965; published 2013 Aug. 22) analyzxed 12 plant RNAviruses to investigatge evoluationary rates and found evidence ofepisodic selection possibly due to shifts between different hostgenotyopes or species. The CRISPR systems and proteins of the instantinvention may be used to tarteg or immunize against such viruses in ahost. For example, the systems of the invention can be used to blockviral RNA expression hence replication. Also, the invention can be usedto target nuclic acids for cleavage as wll as to target expression oractivation. Moreover, the systems of the invention can be multiplexed soas to hit multiple targets or multiple isolate of the same virus.

Ma et al. (Mol Plant. 2015 Aug. 3; 8(8):1274-84. doi:10.1016/j.molp.2015.04.007) reports robust CRISPR-Cas9 vector system,utilizing a plant codon optimized Cas9 gene, for convenient andhigh-efficiency multiplex genome editing in monocot and dicot plants. Maet al. designed PCR-based procedures to rapidly generate multiple sgRNAexpression cassettes, which can be assembled into the binary CRISPR-Cas9vectors in one round of cloning by Golden Gate ligation or GibsonAssembly. With this system, Ma et al. edited 46 target sites in ricewith an average 85.4% rate of mutation, mostly in biallelic andhomozygous status. Ma et al. provide examples of loss-of-function genemutations in T0 rice and T1Arabidopsis plants by simultaneous targetingof multiple (up to eight) members of a gene family, multiple genes in abiosynthetic pathway, or multiple sites in a single gene. Similarly, theCRISPR systems of the instant invention can dffieicnelty targetexpression of multiple genes simultaneously.

Lowder et al. (Plant Physiol. 2015 Aug. 21. pii: pp. 00636.2015) alsodeveloped a CRISPR-Cas9 toolbox enables multiplex genome editing andtranscriptional regulation of expressed, silenced or non-coding genes inplants. This toolbox provides researchers with a protocol and reagentsto quickly and efficiently assemble functional CRISPR-Cas9 T-DNAconstructs for monocots and dicots using Golden Gate and Gateway cloningmethods. It comes with a full suite of capabilities, includingmultiplexed gene editing and transcriptional activation or repression ofplant endogenous genes. T-DNA based transformation technology isfundamental to modern plant biotechnology, genetics, molecular biologyand physiology. As such, we developed a method for the assembly of Cas9(WT, nickase or dCas9) and gRNA(s) into a T-DNA destination-vector ofinterest. The assembly method is based on both Golden Gate assembly andMultiSite Gateway recombination. Three modules are required forassembly. The first module is a Cas9 entry vector, which containspromoterless Cas9 or its derivative genes flanked by attL1 and attR5sites. The second module is a gRNA entry vector which contains entrygRNA expression cassettes flanked by attL5 and attL2 sites. The thirdmodule includes attR1-attR2-containing destination T-DNA vectors thatprovide promoters of choice for Cas9 expression. The toolbox of Lowderet al. may be applied to the CRISPR effector protein system of thepresent invention.

Organisms such as yeast and microalgae are widely used for syntheticbiology. Stovicek et al. (Metab. Eng. Comm., 2015; 2:13 describes genomeediting of industrial yeast, for example, Saccharomyces cerevisae, toefficiently produce robust strains for industrial production. Stovicekused a CRISPR-Cas9 system codon-optimized for yeast to simultaneouslydisrupt both alleles of an endogenous gene and knock in a heterologousgene. Cas9 and gRNA were expressed from genomic or episomal 2-basedvector locations. The authors also showed that gene disruptionefficiency could be improved by optimization of the levels of Cas9 andgRNA expression. Hlavova et al. (Biotechnol. Adv. 2015) discussesdevelopment of species or strains of microalgae using techniques such asCRISPR to target nuclear and chloroplast genes for insertionalmutagenesis and screening. The same plasmids and vectors can be applicedto the CRISPR systems of the instant invention.

Petersen (“Towards precisely glycol engineered plants,” Plant BiotechDenmark Annual meeting 2015, Copenhagen, Denmark) developed a method ofusing CRISPR/Cas9 to engineer genome changes in Arabidopsis, for exampleto glyco engineer Arabidopsis for production of proteins and productshaving desired posttranslational modifications. Hebelstrup et al. (FrontPlant Sci. 2015 Apr. 23; 6:247) outlines inplanta starch bioengineering,providing crops that express starch modifying enzymes and directlyproduce products that normally are made by industrial chemical and/orphysical treatments of starches. The methods of Petersen and Hebelstrupmay be applied to the CRISPR effector protein system of the presentinvention.

Kurthe t al, J Virol. 2012 June; 86(11):6002-9. doi:10.1128/JVI.00436-12. Epub 2012 Mar. 21) developed an RNA virus-basedvector for the introduction of desired traits into grapevine withoutheritable modifications to the genome. The vector provided the abilityto regulate expression of of endogenous genes by virus-induced genesilencing. The CRISPR systems and proteins of the instant invention canbe used to silence genes and proteins without heritable modification tothe genome.

In an embodiment, the plant may be a legume. The present invention mayutilize the herein disclosed CRISP-Cas system for exploring andmodifying, for example, without limitation, soybeans, peas, and peanuts.Curtin et al. provides a toolbox for legume function genomics. (SeeCurtin et al., “A genome engineering toolbox for legume Functionalgenomics,” International Plant and Animal Genome Conference XXII 2014).Curtin used the genetic transformation of CRISPR to knock-out/downsingle copy and duplicated legume genes both in hairy root and wholeplant systems. Some of the target genes were chosen in order to exploreand optimize the features of knock-out/down systems (e.g., phytoenedesaturase), while others were identified by soybean homology toArabidopsis Dicer-like genes or by genome-wide association studies ofnodulation in Medicago. The CRISPR systems and proteins of the instantinvention can be used to knockout/knockdown systems.

Peanut allergies and allergies to legumes generally are a real andserious health concern. The CRISPR effector protein system of thepresent invention can be used to identify and then edit or silence genesencoding allergenic proteins of such legumes. Without limitation as tosuch genes and proteins, Nicolaou et al. identifies allergenic proteinsin peanuts, soybeans, lentils, peas, lupin, green beans, and mung beans.See, Nicolaou et al., Current Opinion in Allergy and Clinical Immunology2011; 11(3):222).

In an advantageous embodiment, the plant may be a tree. The presentinvention may also utilize the herein disclosed CRISPR Cas system forherbaceous systems (see, e.g., Belhaj et al., Plant Methods 9: 39 andHarrison et al., Genes & Development 28: 1859-1872). In a particularlyadvantageous embodiment, the CRISPR Cas system of the present inventionmay target single nucleotide polymorphisms (SNPs) in trees (see, e.g.,Zhou et al., New Phytologist, Volume 208, Issue 2, pages 298-301,October 2015). In the Zhou et al. study, the authors applied a CRISPRCas system in the woody perennial Populus using the 4-coumarate:CoAligase (4CL) gene family as a case study and achieved 100% mutationalefficiency for two 4CL genes targeted, with every transformant examinedcarrying biallelic modifications. In the Zhou et al., study, theCRISPR-Cas9 system was highly sensitive to single nucleotidepolymorphisms (SNPs), as cleavage for a third 4CL gene was abolished dueto SNPs in the target sequence. These methods may be applied to theCRISPR effector protein system of the present invention.

The methods of Zhou et al. (New Phytologist, Volume 208, Issue 2, pages298-301, October 2015) may be applied to the present invention asfollows. Two 4CL genes, 4CL1 and 4CL2, associated with lignin andflavonoid biosynthesis, respectively are targeted for CRISPR-Cas9editing. The Populus tremula x alba clone 717-1B4 routinely used fortransformation is divergent from the genome-sequenced Populustrichocarpa. Therefore, the 4CL1 and 4CL2 gRNAs designed from thereference genome are interrogated with in-house 717 RNA-Seq data toensure the absence of SNPs which could limit Cas efficiency. A thirdgRNA designed for 4CL5, a genome duplicate of 4CL1, is also included.The corresponding 717 sequence harbors one SNP in each allelenear/within the PAM, both of which are expected to abolish targeting bythe 4CL5-gRNA. All three gRNA target sites are located within the firstexon. For 717 transformation, the gRNA is expressed from the MedicagoU6.6 promoter, along with a human codon-optimized Cas under control ofthe CaMV 35S promoter in a binary vector. Transformation with theCas-only vector can serve as a control. Randomly selected 4CL1 and 4CL2lines are subjected to amplicon-sequencing. The data is then processedand biallelic mutations are confirmed in all cases. These methods may beapplied to the CRISPR effector protein system of the present invention.

In plants, pathogens are often host-specific. For example, Fusariumoxysporum f sp. lycopersici causes tomato wilt but attacks only tomato,and F. oxysporum f. dianthii Puccinia graminis f. sp. tritici attacksonly wheat. Plants have existing and induced defenses to resist mostpathogens. Mutations and recombination events across plant generationslead to genetic variability that gives rise to susceptibility,especially as pathogens reproduce with more frequency than plants. Inplants there can be non-host resistance, e.g., the host and pathogen areincompatible. There can also be Horizontal Resistance, e.g., partialresistance against all races of a pathogen, typically controlled by manygenes and Vertical Resistance, e.g., complete resistance to some racesof a pathogen but not to other races, typically controlled by a fewgenes. In a Gene-for-Gene level, plants and pathogens evolve together,and the genetic changes in one balance changes in other. Accordingly,using Natural Variability, breeders combine most useful genes for Yield,Quality, Uniformity, Hardiness, Resistance. The sources of resistancegenes include native or foreign Varieties, Heirloom Varieties, WildPlant Relatives, and Induced Mutations, e.g., treating plant materialwith mutagenic agents. Using the present invention, plant breeders areprovided with a new tool to induce mutations. Accordingly, one skilledin the art can analyze the genome of sources of resistance genes, and inVarieties having desired characteristics or traits employ the presentinvention to induce the rise of resistance genes, with more precisionthan previous mutagenic agents and hence accelerate and improve plantbreeding programs.

Aside from the plants otherwise discussed herein and above, engineeredplants modified by the effector protein and suitable guide, and progenythereof, as provided. These may include disease or drought resistantcrops, such as wheat, barley, rice, soybean or corn; plants modified toremove or reduce the ability to self-pollinate (but which can instead,optionally, hybridise instead); and allergenic foods such as peanuts andnuts where the immunogenic proteins have been disabled, destroyed ordisrupted by targeting via a effector protein and suitable guide.

With respect to general information on CRISPR-Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, AAV, and making and usingthereof, including as to amounts and formulations, all useful in thepractice of the instant invention, reference is made to: U.S. Pat. Nos.8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616, 8,895,308,8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945 and8,697,359; US Patent Publications US 2014-0310830 (U.S. application Ser.No. 14/105,031), US 2014-0287938 A1 (U.S. application Ser. No.14/213,991), US 2014-0273234 A1 (U.S. application Ser. No. 14/293,674),US2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US2014-0273231 (U.S. application Ser. No. 14/259,420), US 2014-0256046 A1(U.S. application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S.application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. applicationSer. No. 14/222,930), US 2014-0242699 A1 (U.S. application Ser. No.14/183,512), US 2014-0242664 A1 (U.S. application Ser. No. 14/104,990),US 2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US2014-0227787 A1 (U.S. application Ser. No. 14/256,912), US 2014-0189896A1 (U.S. application Ser. No. 14/105,035), US 2014-0186958 (U.S.application Ser. No. 14/105,017), US 2014-0186919 A1 (U.S. applicationSer. No. 14/104,977), US 2014-0186843 A1 (U.S. application Ser. No.14/104,900), US 2014-0179770 A1 (U.S. application Ser. No. 14/104,837)and US 2014-0179006 A1 (U.S. application Ser. No. 14/183,486), US2014-0170753 (U.S. application Ser. No. 14/183,429); European Patents EP2 784 162 B1 and EP 2 771 468 B1; European Patent Applications EP 2 771468 (EP13818570.7), EP 2 764 103 (EP13824232.6), and EP 2 784 162(EP14170383.5); and PCT Patent Publications PCT Patent Publications WO2014/093661 (PCT/US2013/074743), WO 2014/093694 (PCT/US2013/074790), WO2014/093595 (PCT/US2013/074611), WO 2014/093718 (PCT/US2013/074825), WO2014/093709 (PCT/US2013/074812), WO 2014/093622 (PCT/US2013/074667), WO2014/093635 (PCT/US2013/074691), WO 2014/093655 (PCT/US2013/074736), WO2014/093712 (PCT/US2013/074819), WO 2014/093701 (PCT/US2013/074800), WO2014/018423 (PCT/US2013/051418), WO 2014/204723 (PCT/US2014/041790), WO2014/204724 (PCT/US2014/041800), WO 2014/204725 (PCT/US2014/041803), WO2014/204726 (PCT/US2014/041804), WO 2014/204727 (PCT/US2014/041806), WO2014/204728 (PCT/US2014/041808), WO 2014/204729 (PCT/US2014/041809).Reference is also made to U.S. provisional patent applications61/758,468; 61/802,174; 61/806,375; 61/814,263; 61/819,803 and61/828,130, filed on Jan. 30, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr.20, 2013; May 6, 2013 and May 28, 2013 respectively. Reference is alsomade to U.S. provisional patent application 61/836,123, filed on Jun.17, 2013. Reference is additionally made to U.S. provisional patentapplications 61/835,931, 61/835,936, 61/836,127, 61/836,101, 61/836,080and 61/835,973, each filed Jun. 17, 2013. Further reference is made toU.S. provisional patent applications 61/862,468 and 61/862,355 filed onAug. 5, 2013; 61/871,301 filed on Aug. 28, 2013; 61/960,777 filed onSep. 25, 2013 and 61/961,980 filed on Oct. 28, 2013. Reference is yetfurther made to: PCT Patent applications Nos: PCT/US2014/041803,PCT/US2014/041800, PCT/US2014/041809, PCT/US2014/041804 andPCT/US2014/041806, each filed Jun. 10, 2014 6 Oct. 2014;PCT/US2014/041808 filed Jun. 11, 2014; and PCT/US2014/62558 filed Oct.28, 2014, and U.S. Provisional Patent Application Ser. Nos. 61/915,150,61/915,301, 61/915,267 and 61/915,260, each filed Dec. 12, 2013;61/757,972 and 61/768,959, filed on Jan. 29, 2013 and Feb. 25, 2013;61/835,936, 61/836,127, 61/836,101, 61/836,080, 61/835,973, and61/835,931, filed Jun. 17, 2013; 62/010,888 and 62/010,879, both filedJun. 11, 2014; 62/010,329 and 62/010,441, each filed Jun. 10, 2014;61/939,228 and 61/939,242, each filed Feb. 12, 2014; 61/980,012, filedApr. 15, 2014; 62/038,358, filed Aug. 17, 2014; 62/054,490, 62/055,484,62/055,460 and 62/055,487, each filed Sep. 25, 2014; and 62/069,243,filed Oct. 27, 2014. Reference is also made to U.S. provisional patentapplications Nos. 62/055,484, 62/055,460, and 62/055,487, filed Sep. 25,2014; U.S. provisional patent application 61/980,012, filed Apr. 15,2014; and U.S. provisional patent application 61/939,242 filed Feb. 12,2014. Reference is made to PCT application designating, inter alia, theUnited States, application No. PCT/US14/41806, filed Jun. 10, 2014.Reference is made to U.S. provisional patent application 61/930,214filed on Jan. 22, 2014. Reference is made to U.S. provisional patentapplications 61/915,251; 61/915,260 and 61/915,267, each filed on Dec.12, 2013. Reference is made to US provisional patent application U.S.Ser. No. 61/980,012 filed Apr. 15, 2014. Reference is made to PCTapplication designating, inter alia, the United States, application No.PCT/US14/41806, filed Jun. 10, 2014. Reference is made to U.S.provisional patent application 61/930,214 filed on Jan. 22, 2014.Reference is made to U.S. provisional patent applications 61/915,251;61/915,260 and 61/915,267, each filed on Dec. 12, 2013.

Mention is also made of U.S. application 62/091,455, filed, 12 Dec.2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708, 24Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,462,12 Dec. 2014, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; U.S.application 62/096,324, 23 Dec. 2014, DEAD GUIDES FOR CRISPRTRANSCRIPTION FACTORS; U.S. application 62/091,456, 12 Dec. 2014,ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; U.S.application 62/091,461, 12 Dec. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOMEEDITING AS TO HEMATOPOETIC STEM CELLS (HSCs); U.S. application62/094,903, 19 Dec. 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRANDBREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURESEQUENCING; U.S. application 62/096,761, 24 Dec. 2014, ENGINEERING OFSYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCEMANIPULATION; U.S. application 62/098,059, 30 Dec. 2014, RNA-TARGETINGSYSTEM; U.S. application 62/096,656, 24 Dec. 2014, CRISPR HAVING ORASSOCIATED WITH DESTABILIZATION DOMAINS; U.S. application 62/096,697, 24Dec. 2014, CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. application62/098,158, 30 Dec. 2014, ENGINEERED CRISPR COMPLEX INSERTIONALTARGETING SYSTEMS; U.S. application 62/151,052, 22 Apr. 2015, CELLULARTARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; U.S. application62/054,490, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS ANDDISEASES USING PARTICLE DELIVERY COMPONENTS; U.S. application62/055,484, 25 Sep. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/087,537, 4 Dec. 2014, SYSTEMS, METHODS AND COMPOSITIONSFOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;U.S. application 62/054,651, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELINGCOMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. application62/067,886, 23 Oct. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OFMULTIPLE CANCER MUTATIONS IN VIVO; U.S. application 62/054,675, 24 Sep.2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS INNEURONAL CELLS/TISSUES; U.S. application62/054,528, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN IMMUNE DISEASES OR DISORDERS;U.S. application 62/055,454, 25 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING CELL PENETRATION PEPTIDES (CPP); U.S.application 62/055,460, 25 Sep. 2014, MULTIFUNCTIONAL-CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; U.S.application 62/087,475, 4 Dec. 2014, FUNCTIONAL SCREENING WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/055,487, 25 Sep.2014, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;U.S. application 62/087,546, 4 Dec. 2014, MULTIFUNCTIONAL CRISPRCOMPLEXES AND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES;and U.S. application 62/098,285, 30 Dec. 2014, CRISPR MEDIATED IN VIVOMODELING AND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appln citeddocuments”) and all documents cited or referenced in the appln citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appln cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Also with respect to general information on CRISPR-Cas Systems, mentionis made of the following (also hereby incorporated herein by reference):

-   Multiplex genome engineering using CRISPR/Cas systems. Cong, L.,    Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D.,    Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science February    15; 339(6121):819-23 (2013);-   RNA-guided editing of bacterial genomes using CRISPR-Cas systems.    Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A. Nat Biotechnol    March; 31(3):233-9 (2013);-   One-Step Generation of Mice Carrying Mutations in Multiple Genes by    CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H., Shivalila    C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R. Cell May 9;    153(4):910-8 (2013);-   Optical control of mammalian endogenous transcription and epigenetic    states. Konermann S, Brigham M D, Trevino A E, Hsu P D, Heidenreich    M, Cong L, Platt R J, Scott D A, Church G M, Zhang F. Nature. August    22; 500(7463):472-6. doi: 10.1038/Nature12466. Epub 2013 Aug. 23    (2013);-   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing    Specificity. Ran, F A., Hsu, P D., Lin, C Y., Gootenberg, J S.,    Konermann, S., Trevino, A E., Scott, D A., Inoue, A., Matoba, S.,    Zhang, Y., & Zhang, F. Cell August 28. pii: S0092-8674(13)01015-5    (2013-A);-   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,    Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V.,    Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L    A., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013);-   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu, P    D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature    Protocols November; 8(11):2281-308 (2013-B);-   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem,    O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson,    T., Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F.    Science December 12. (2013). [Epub ahead of print];-   Crystal structure of cas9 in complex with guide RNA and target DNA.    Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S., Shehata, S I.,    Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell February 27,    156(5):935-49 (2014);-   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian    cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon D    B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch R.,    Zhang F., Sharp P A. Nat Biotechnol. April 20. doi: 10.1038/nbt.2889    (2014);-   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.    Platt R J, Chen S, Zhou Y, Yim M J, Swiech L, Kempton H R, Dahlman J    E, Parnas O, Eisenhaure T M, Jovanovic M, Graham D B, Jhunjhunwala    S, Heidenreich M, Xavier R J, Langer R, Anderson D G, Hacohen N,    Regev A, Feng G, Sharp P A, Zhang F. Cell 159(2): 440-455 DOI:    10.1016/j.cell.2014.09.014(2014);-   Development and Applications of CRISPR-Cas9 for Genome Engineering,    Hsu P D, Lander E S, Zhang F., Cell. June 5; 157(6):1262-78 (2014).-   Genetic screens in human cells using the CRISPR/Cas9 system, Wang T,    Wei J J, Sabatini D M, Lander E S., Science. January 3; 343(6166):    80-84. doi:10.1126/science.1246981 (2014);-   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated    gene inactivation, Doench J G, Hartenian E, Graham D B, Tothova Z,    Hegde M, Smith I, Sullender M, Ebert B L, Xavier R J, Root D E.,    (published online 3 Sep. 2014) Nat Biotechnol. December;    32(12):1262-7 (2014);-   In vivo interrogation of gene function in the mammalian brain using    CRISPR-Cas9, Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y,    Trombetta J, Sur M, Zhang F., (published online 19 Oct. 2014) Nat    Biotechnol. January; 33(1):102-6 (2015);-   Genome-scale transcriptional activation by an engineered CRISPR-Cas9    complex, Konermann S, Brigham M D, Trevino A E, Joung J, Abudayyeh O    O, Barcena C, Hsu P D, Habib N, Gootenberg J S, Nishimasu H, Nureki    O, Zhang F., Nature. January 29; 517(7536):583-8 (2015).-   A split-Cas9 architecture for inducible genome editing and    transcription modulation, Zetsche B, Volz S E, Zhang F., (published    online 2 Feb. 2015) Nat Biotechnol. February; 33(2):139-42 (2015);-   Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and    Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi X,    Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F, Sharp P A.    Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen in mouse), and-   In vivo genome editing using Staphylococcus aureus Cas9, Ran F A,    Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche B,    Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang F.,    (published online 1 Apr. 2015), Nature. April 9;    520(7546):186-91(2015).-   Shalem et al., “High-throughput functional genomics using    CRISPR-Cas9,” Nature Reviews Genetics 16, 299-311 (May 2015).-   Xu et al., “Sequence determinants of improved CRISPR sgRNA design,”    Genome Research 25, 1147-1157 (August 2015).-   Parnas et al., “A Genome-wide CRISPR Screen in Primary Immune Cells    to Dissect Regulatory Networks,” Cell 162, 675-686 (Jul. 30, 2015).-   Ramanan et al., CRISPR/Cas9 cleavage of viral DNA efficiently    suppresses hepatitis B virus,” Scientific Reports 5:10833. doi:    10.1038/srep10833 (Jun. 2, 2015)-   Nishimasu et al., “Crystal Structure of Staphylococcus aureus Cas9,”    Cell 162, 1113-1126 (Aug. 27, 2015)-   Zetsche et al. (2015), “Cpf1 is a single RNA-guided endonuclease of    a class 2 CRISPR-Cas system,” Cell 163, 759-771 (Oct. 22, 2015) doi:    10.1016/j.cell.2015.09.038. Epub Sep. 25, 2015-   Shmakov et al. (2015), “Discovery and Functional Characterization of    Diverse Class 2 CRISPR-Cas Systems,” Molecular Cell 60, 385-397    (Nov. 5, 2015) doi: 10.1016/j.molcel.2015.10.008. Epub Oct. 22, 2015-   Gao et al, “Engineered Cpf1 Enzymes with Altered PAM Specificities,”    bioRxiv 091611; doi: http://dx.doi.org/10.1101/091611 Epub Dec. 4,    2016    each of which is incorporated herein by reference, may be considered    in the practice of the instant invention, and discussed briefly    below:    -   Cong et al. engineered type II CRISPR-Cas systems for use in        eukaryotic cells based on both Streptococcus thermophilus Cas9        and also Streptococcus pyogenes Cas9 and demonstrated that Cas9        nucleases can be directed by short RNAs to induce precise        cleavage of DNA in human and mouse cells. Their study further        showed that Cas9 as converted into a nicking enzyme can be used        to facilitate homology-directed repair in eukaryotic cells with        minimal mutagenic activity. Additionally, their study        demonstrated that multiple guide sequences can be encoded into a        single CRISPR array to enable simultaneous editing of several at        endogenous genomic loci sites within the mammalian genome,        demonstrating easy programmability and wide applicability of the        RNA-guided nuclease technology. This ability to use RNA to        program sequence specific DNA cleavage in cells defined a new        class of genome engineering tools. These studies further showed        that other CRISPR loci are likely to be transplantable into        mammalian cells and can also mediate mammalian genome cleavage.        Importantly, it can be envisaged that several aspects of the        CRISPR-Cas system can be further improved to increase its        efficiency and versatility.    -   Jiang et al. used the clustered, regularly interspaced, short        palindromic repeats (CRISPR)-associated Cas9 endonuclease        complexed with dual-RNAs to introduce precise mutations in the        genomes of Streptococcus pneumoniae and Escherichia coli. The        approach relied on dual-RNA:Cas9-directed cleavage at the        targeted genomic site to kill unmutated cells and circumvents        the need for selectable markers or counter-selection systems.        The study reported reprogramming dual-RNA:Cas9 specificity by        changing the sequence of short CRISPR RNA (crRNA) to make        single- and multinucleotide changes carried on editing        templates. The study showed that simultaneous use of two crRNAs        enabled multiplex mutagenesis. Furthermore, when the approach        was used in combination with recombineering, in S. pneumoniae,        nearly 100% of cells that were recovered using the described        approach contained the desired mutation, and in E. coli, 65%        that were recovered contained the mutation.    -   Wang et al. (2013) used the CRISPR/Cas system for the one-step        generation of mice carrying mutations in multiple genes which        were traditionally generated in multiple steps by sequential        recombination in embryonic stem cells and/or time-consuming        intercrossing of mice with a single mutation. The CRISPR/Cas        system will greatly accelerate the in vivo study of functionally        redundant genes and of epistatic gene interactions.    -   Konermann et al. (2013) addressed the need in the art for        versatile and robust technologies that enable optical and        chemical modulation of DNA-binding domains based CRISPR Cas9        enzyme and also Transcriptional Activator Like Effectors    -   Ran et al. (2013-A) described an approach that combined a Cas9        nickase mutant with paired guide RNAs to introduce targeted        double-strand breaks. This addresses the issue of the Cas9        nuclease from the microbial CRISPR-Cas system being targeted to        specific genomic loci by a guide sequence, which can tolerate        certain mismatches to the DNA target and thereby promote        undesired off-target mutagenesis. Because individual nicks in        the genome are repaired with high fidelity, simultaneous nicking        via appropriately offset guide RNAs is required for        double-stranded breaks and extends the number of specifically        recognized bases for target cleavage. The authors demonstrated        that using paired nicking can reduce off-target activity by 50-        to 1,500-fold in cell lines and to facilitate gene knockout in        mouse zygotes without sacrificing on-target cleavage efficiency.        This versatile strategy enables a wide variety of genome editing        applications that require high specificity.    -   Hsu et al. (2013) characterized SpCas9 targeting specificity in        human cells to inform the selection of target sites and avoid        off-target effects. The study evaluated >700 guide RNA variants        and SpCas9-induced indel mutation levels at >100 predicted        genomic off-target loci in 293T and 293FT cells. The authors        that SpCas9 tolerates mismatches between guide RNA and target        DNA at different positions in a sequence-dependent manner,        sensitive to the number, position and distribution of        mismatches. The authors further showed that SpCas9-mediated        cleavage is unaffected by DNA methylation and that the dosage of        SpCas9 and sgRNA can be titrated to minimize off-target        modification. Additionally, to facilitate mammalian genome        engineering applications, the authors reported providing a        web-based software tool to guide the selection and validation of        target sequences as well as off-target analyses.    -   Ran et al. (2013-B) described a set of tools for Cas9-mediated        genome editing via non-homologous end joining (NHEJ) or        homology-directed repair (HDR) in mammalian cells, as well as        generation of modified cell lines for downstream functional        studies. To minimize off-target cleavage, the authors further        described a double-nicking strategy using the Cas9 nickase        mutant with paired guide RNAs. The protocol provided by the        authors experimentally derived guidelines for the selection of        target sites, evaluation of cleavage efficiency and analysis of        off-target activity. The studies showed that beginning with        target design, gene modifications can be achieved within as        little as 1-2 weeks, and modified clonal cell lines can be        derived within 2-3 weeks.    -   Shalem et al. described a new way to interrogate gene function        on a genome-wide scale. Their studies showed that delivery of a        genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted        18,080 genes with 64,751 unique guide sequences enabled both        negative and positive selection screening in human cells. First,        the authors showed use of the GeCKO library to identify genes        essential for cell viability in cancer and pluripotent stem        cells. Next, in a melanoma model, the authors screened for genes        whose loss is involved in resistance to vemurafenib, a        therapeutic that inhibits mutant protein kinase BRAF. Their        studies showed that the highest-ranking candidates included        previously validated genes NF1 and MED12 as well as novel hits        NF2, CUL3, TADA2B, and TADA1. The authors observed a high level        of consistency between independent guide RNAs targeting the same        gene and a high rate of hit confirmation, and thus demonstrated        the promise of genome-scale screening with Cas9.    -   Nishimasu et al. reported the crystal structure of Streptococcus        pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°        resolution. The structure revealed a bilobed architecture        composed of target recognition and nuclease lobes, accommodating        the sgRNA:DNA heteroduplex in a positively charged groove at        their interface. Whereas the recognition lobe is essential for        binding sgRNA and DNA, the nuclease lobe contains the HNH and        RuvC nuclease domains, which are properly positioned for        cleavage of the complementary and non-complementary strands of        the target DNA, respectively. The nuclease lobe also contains a        carboxyl-terminal domain responsible for the interaction with        the protospacer adjacent motif (PAM). This high-resolution        structure and accompanying functional analyses have revealed the        molecular mechanism of RNA-guided DNA targeting by Cas9, thus        paving the way for the rational design of new, versatile        genome-editing technologies.    -   Wu et al. mapped genome-wide binding sites of a catalytically        inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with        single guide RNAs (sgRNAs) in mouse embryonic stem cells        (mESCs). The authors showed that each of the four sgRNAs tested        targets dCas9 to between tens and thousands of genomic sites,        frequently characterized by a 5-nucleotide seed region in the        sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin        inaccessibility decreases dCas9 binding to other sites with        matching seed sequences; thus 70% of off-target sites are        associated with genes. The authors showed that targeted        sequencing of 295 dCas9 binding sites in mESCs transfected with        catalytically active Cas9 identified only one site mutated above        background levels. The authors proposed a two-state model for        Cas9 binding and cleavage, in which a seed match triggers        binding but extensive pairing with target DNA is required for        cleavage.    -   Platt et al. established a Cre-dependent Cas9 knockin mouse. The        authors demonstrated in vivo as well as ex vivo genome editing        using adeno-associated virus (AAV)-, lentivirus-, or        particle-mediated delivery of guide RNA in neurons, immune        cells, and endothelial cells.    -   Hsu et al. (2014) is a review article that discusses generally        CRISPR-Cas9 history from yogurt to genome editing, including        genetic screening of cells.    -   Wang et al. (2014) relates to a pooled, loss-of-function genetic        screening approach suitable for both positive and negative        selection that uses a genome-scale lentiviral single guide RNA        (sgRNA) library.    -   Doench et al. created a pool of sgRNAs, tiling across all        possible target sites of a panel of six endogenous mouse and        three endogenous human genes and quantitatively assessed their        ability to produce null alleles of their target gene by antibody        staining and flow cytometry. The authors showed that        optimization of the PAM improved activity and also provided an        on-line tool for designing sgRNAs.    -   Swiech et al. demonstrate that AAV-mediated SpCas9 genome        editing can enable reverse genetic studies of gene function in        the brain.    -   Konermann et al. (2015) discusses the ability to attach multiple        effector domains, e.g., transcriptional activator, functional        and epigenomic regulators at appropriate positions on the guide        such as stem or tetraloop with and without linkers.    -   Zetsche et al. demonstrates that the Cas9 enzyme can be split        into two and hence the assembly of Cas9 for activation can be        controlled.    -   Chen et al. relates to multiplex screening by demonstrating that        a genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes        regulating lung metastasis.    -   Ran et al. (2015) relates to SaCas9 and its ability to edit        genomes and demonstrates that one cannot extrapolate from        biochemical assays. Shalem et al. (2015) described ways in which        catalytically inactive Cas9 (dCas9) fusions are used to        synthetically repress (CRISPRi) or activate (CRISPRa)        expression, showing. advances using Cas9 for genome-scale        screens, including arrayed and pooled screens, knockout        approaches that inactivate genomic loci and strategies that        modulate transcriptional activity.    -   Shalem et al. (2015) described ways in which catalytically        inactive Cas9 (dCas9) fusions are used to synthetically repress        (CRISPRi) or activate (CRISPRa) expression, showing. advances        using Cas9 for genome-scale screens, including arrayed and        pooled screens, knockout approaches that inactivate genomic loci        and strategies that modulate transcriptional activity.    -   Xu et al. (2015) assessed the DNA sequence features that        contribute to single guide RNA (sgRNA) efficiency in        CRISPR-based screens. The authors explored efficiency of        CRISPR/Cas9 knockout and nucleotide preference at the cleavage        site. The authors also found that the sequence preference for        CRISPRi/a is substantially different from that for CRISPR/Cas9        knockout.    -   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9        libraries into dendritic cells (DCs) to identify genes that        control the induction of tumor necrosis factor (Tnf) by        bacterial lipopolysaccharide (LPS). Known regulators of Tlr4        signaling and previously unknown candidates were identified and        classified into three functional modules with distinct effects        on the canonical responses to LPS.    -   Ramanan et al (2015) demonstrated cleavage of viral episomal DNA        (cccDNA) in infected cells. The HBV genome exists in the nuclei        of infected hepatocytes as a 3.2 kb double-stranded episomal DNA        species called covalently closed circular DNA (cccDNA), which is        a key component in the HBV life cycle whose replication is not        inhibited by current therapies. The authors showed that sgRNAs        specifically targeting highly conserved regions of HBV robustly        suppresses viral replication and depleted cccDNA.    -   Nishimasu et al. (2015) reported the crystal structures of        SaCas9 in complex with a single guide RNA (sgRNA) and its        double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and        the 5′-TTGGGT-3′ PAM. A structural comparison of SaCas9 with        SpCas9 highlighted both structural conservation and divergence,        explaining their distinct PAM specificities and orthologous        sgRNA recognition.    -   Zetsche et al. (2015) reported the characterization of Cpf1, a        putative class 2 CRISPR effector. It was demonstrated that Cpf1        mediates robust DNA interference with features distinct from        Cas9. Identifying this mechanism of interference broadens our        understanding of CRISPR-Cas systems and advances their genome        editing applications.    -   Shmakov et al. (2015) reported the characterization of three        distinct Class 2 CRISPR-Cas systems. The effectors of two of the        identified systems, C2c1 and C2c3, contain RuvC like        endonuclease domains distantly related to Cpf1. The third        system, C2c2, contains an effector with two predicted HEPN RNase        domains.    -   Gao et al. (2016) reported using a structure-guided saturation        mutagenesis screen to increase the targeting range of Cpf1.        AsCpf1 variants were engineered with the mutations S542R/K607R        and S542R/K548V/N552R that can cleave target sites with        TYCV/CCCC and TATV PAMs, respectively, with enhanced activities        in vitro and in human cells.

Also, “Dimeric CRISPR RNA-guided FokI nucleases for highly specificgenome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter,Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin,Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77(2014), relates to dimeric RNA-guided FokI Nucleases that recognizeextended sequences and can edit endogenous genes with high efficienciesin human cells.

In addition, mention is made of PCT application PCT/US 14/70057,Attorney Reference 47627.99.2060 and BI-2013/107 entitiled “DELIVERY,USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS ANDCOMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLEDELIVERY COMPONENTS (claiming priority from one or more or all of USprovisional patent applications: 62/054,490, filed Sep. 24, 2014;62/010,441, filed Jun. 10, 2014; and 61/915,118, 61/915,215 and61/915,148, each filed on Dec. 12, 2013) (“the Particle Delivery PCT”),incorporated herein by reference, with respect to a method of preparingan sgRNA-and-Cas9 protein containing particle comprising admixing amixture comprising an sgRNA and Cas9 protein (and optionally HDRtemplate) with a mixture comprising or consisting essentially of orconsisting of surfactant, phospholipid, biodegradable polymer,lipoprotein and alcohol; and particles from such a process. For example,wherein Cas9 protein and sgRNA were mixed together at a suitable, e.g.,3:1 to 1:3 or 2:1 to 1:2 or 1:1 molar ratio, at a suitable temperature,e.g., 15-30C, e.g., 20-25C, e.g., room temperature, for a suitable time,e.g., 15-45, such as 30 minutes, advantageously in sterile, nucleasefree buffer, e.g., 1×PBS. Separately, particle components such as orcomprising: a surfactant, e.g., cationic lipid, e.g.,1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); phospholipid, e.g.,dimyristoylphosphatidylcholine (DMPC); biodegradable polymer, such as anethylene-glycol polymer or PEG, and a lipoprotein, such as a low-densitylipoprotein, e.g., cholesterol were dissolved in an alcohol,advantageously a C₁₋₆ alkyl alcohol, such as methanol, ethanol,isopropanol, e.g., 100% ethanol. The two solutions were mixed togetherto form particles containing the Cas9-sgRNA complexes. Accordingly,sgRNA may be pre-complexed with the Cas9 protein, before formulating theentire complex in a particle. Formulations may be made with a differentmolar ratio of different components known to promote delivery of nucleicacids into cells (e.g. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol) For example DOTAP:DMPC:PEG:CholesterolMolar Ratios may be DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; or DOTAP90, DMPC 0, PEG 10, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 5,Cholesterol 5. DOTAP 100, DMPC 0, PEG 0, Cholesterol 0. That applicationaccordingly comprehends admixing sgRNA, Cas9 protein and components thatform a particle; as well as particles from such admixing. Aspects of theinstant invention can involve particles; for example, particles using aprocess analogous to that of the Particle Delivery PCT, e.g., byadmixing a mixture comprising sgRNA and/or Cas9 as in the instantinvention and components that form a particle, e.g., as in the ParticleDelivery PCT, to form a particle and particles from such admixing (or,of course, other particles involving sgRNA and/or Cas9 as in the instantinvention).

Viral Detection, Monitoring, and Diagnosis

Treating and surveilling viral infections remains a scientificchallenge. In the past 50 years, the world has produced 90 clinicallyapproved antiviral drugs, but these drugs are only treat 9 viraldiseases, less than 10% of the 130 viruses known to be human pathogens(2). Vaccines have emerged as the predominant approach to combatingviral diseases, but only 16 viruses are covered by FDA-approvedvaccines. Viral nucleic acid diagnostics are equally sparse, as only 11viruses currently have an FDA-approved nucleic acid diagnostic. Thus,there remains a pressing need both to detect viruses and to developnovel antiviral therapies, especially in light of recent major outbreaksacross the globe.

Viral evolution exacerbates the challenges already associated withantiviral therapy and viral detection. Antiviral therapy drivesselection leading to resistant viral genomes, and mutations in the viralgenome can decrease the sensitivity of nucleic acid diagnostics. Tocounteract viral evolution, we need to better understand the process bywhich antiviral therapy resistance evolves. Concomitantly, we needtherapeutic approaches that can be easily adapted if a virus evolvesresistance, paired with diagnostics to detect resistance mutations whichcould emerge over time.

In certain aspects of the invention, a sequence-specific nuclease, suchas a CRISPR effector protein, has the potential to solve thesetherapeutic and diagnostic challenges. CRISPR-based technologies haverevolutionized many areas of biology, including genome editing,high-throughput screening, and chromosome imaging.

The sequence-specific nature of CRISPR/Cas system cleavage naturallylends itself to therapeutic and diagnostic applications. The presentinventors have found that Cas13 is able to target the RNA of Lymphocyticchoriomeningitis virus (LCMV), a genetic relative of Lassa virus whichinfects tens of thousands of West Africans annually, thereby inhibitingits replication. In addition to therapy, Cas13 is uniquely poised forviral detection using the recently published Specific High sensitivityEnzymatic Reporter unlocking (SHERLOCK) platform (Grootenberg et al.(2017), “Nucleic acid detection with CRISPR-Cas13/C2c2”, Science,356(6336):438-442). SHERLOCK can detect RNA with single moleculesensitivity and has the specificity to identify single nucleotidepolymorphisms.

In order to realize the potential of Cas13-based therapeutics anddiagnostics, we must uncover mechanisms surrounding Cas13 cleavage andviral evolution. An important scientific question is how target RNAsequence, secondary structure, and gene function influenceCas13-mediated inhibition of viral replication. Secondarily, virusesrapidly generate mutations, allowing them to escape many antiviralselective pressures, possibly including Cas13 targeting.

In certain aspects, the present invention relates to a Class 2, type VICRISPR system-based detection and therapeutic technologies, such asCas13-based detection and therapeutic technologies to understand andcounteract the evolution of viruses that can cause infectious outbreaks,such as in humans. In certain aspects, the invention relates to methodsfor determining how viral genomes evolve in response to Class 2, type VICRISPR system, such as Cas13, targeting. In certain aspects, theinvention relates to methods for preventing guide RNA targetingresistance from evolving, such as by using multiplexed therapies. Incertain aspects, the invention relates to methods for rapidlyidentifying viral pathogens and/or determining if they have acquired anyundesired mutations.

In one aspect, the invention provides a nucleic acid detection system(suitable for the detection or diagnostic methods as described hereinelsewhere, such as suitable for a companion or complementary diagnosticmethod according to certain aspects of the invention as describedherein) comprising: a CRISPR system comprising an effector protein andone or more guide RNAs designed to bind to corresponding targetmolecules; a RNA-based masking construct; and optionally, nucleic acidamplification reagents to amplify target RNA molecules in a sample. Inanother aspect, the embodiments provide a polypeptide detection systemcomprising: a CRISPR system comprising an effector protein and one ormore guide RNAs designed to bind a trigger RNA, a RNA-based maskingconstruct; and one or more detection aptamers comprising a masked RNApolymerase promoter binding site or a masked primer binding site. Anexemplary method is for example described in Grootenberg et al. (2017),“Nucleic acid detection with CRISPR-Cas13/C2c2”, Science,356(6336):438-442, incorporated herein by reference in its entirety.

As used herein, a “companion diagnostic” or “companion diagnosticmethod” refers to a diagnostic test used as a companion to a therapeuticdrug to determine its applicability to a specific person. Companiondiagnostics are typically co-developed with drugs to aid in selecting orexcluding patient groups for treatment with that particular drug on thebasis of their biological characteristics that determine responders andnon-responders to the therapy. A companion diagnostic may be a medicaldevice, often an in vitro device, which provides information that ispreferably essential for the safe and effective use of a correspondingdrug or biological product. The test helps a health care professionaldetermine whether a particular therapeutic product's benefits topatients will outweigh any potential serious side effects or risks.Companion diagnostics can for instance identify patients who are mostlikely to benefit from a particular therapeutic product; identifypatients likely to be at increased risk for serious side effects as aresult of treatment with a particular therapeutic product; or monitorresponse to treatment with a particular therapeutic product for thepurpose of adjusting treatment to achieve improved safety oreffectiveness.

In further embodiments, the system may further comprise nucleic acidamplification reagents. The nucleic acid amplification reagents maycomprise a primer comprising an RNA polymerase promoter. In certainembodiments, sample nucleic acids are amplified to obtain a DNA templatecomprising an RNA polymerase promoter, whereby a target RNA molecule maybe generated by transcription. The nucleic acid may be DNA and amplifiedby any method described herein. The nucleic acid may be RNA andamplified by a reverse transcription method as described herein. Theaptamer sequence may be amplified upon unmasking of the primer bindingsite, whereby a trigger RNA is transcribed from the amplified DNAproduct. The target molecule may be a target DNA and the system mayfurther comprises a primer that binds the target DNA and comprises a RNApolymerase promoter.

In another embodiment, the one or more guide RNAs are designed to detecta single nucleotide polymorphism, splice variant of a transcript, or aframeshift mutation in a target RNA or DNA.

In other embodiments, the one or more guide RNAs are designed to bind toone or more target molecules that are diagnostic for a (viral) diseaseor disease state/grade.

In other embodiments of the invention, the RNA-based masking constructsuppresses generation of a detectable positive signal or the RNA-basedmasking construct suppresses generation of a detectable positive signalby masking the detectable positive signal, or generating a detectablenegative signal instead, or the RNA-based masking construct comprises asilencing RNA that suppresses generation of a gene product encoded by areporting construct, wherein the gene product generates the detectablepositive signal when expressed.

In further embodiments, the RNA-based masking construct is a ribozymethat generates the negative detectable signal, and wherein the positivedetectable signal is generated when the ribozyme is deactivated, or theribozyme converts a substrate to a first color and wherein the substrateconverts to a second color when the ribozyme is deactivated.

In other embodiments, the RNA-based masking agent is a RNA aptamer, orthe aptamer sequesters an enzyme, wherein the enzyme generates adetectable signal upon release from the aptamer by acting upon asubstrate, or the aptamer sequesters a pair of agents that when releasedfrom the aptamers combine to generate a detectable signal.

In another embodiment, the RNA-based masking construct comprises a RNAoligonucleotide to which a detectable ligand and a masking component areattached. In another embodiment, the detectable ligand is a fluorophoreand the masking component is a quencher molecule, or the reagents toamplify target RNA molecules such as, but not limited to, NASBA or RPAreagents.

In another aspect, the invention provides a diagnostic device comprisingone or more individual discrete volumes, each individual discretevolumes comprising a CRISPR effector protein, one or more guide RNAsdesigned to bind to corresponding target molecule, a RNA-based maskingconstruct, and optionally further comprise nucleic acid amplificationreagents.

In another aspect, the invention provides a diagnostic device comprisingone or more individual discrete volumes, each individual discrete volumecomprising a CRISPR effector protein, one or more guide RNAs designed tobind to a trigger RNA, one or more detection aptamers comprising amasked RNA polymerase promoter binding site or a masked primer bindingsite, and optionally further comprising nucleic acid amplificationreagents.

In some embodiments, the individual discrete volumes are droplets, orthe individual discrete volumes are defined on a solid substrate, or theindividual discrete volumes are microwells, or the individual discretevolumes are spots defined on a substrate, such as a paper substrate.

In another aspect, the invention provides a method for detecting targetRNAs in samples, comprising: distributing a sample or set of samplesinto one or more individual discrete volumes, the individual discretevolumes comprising a CRISPR system comprising an effector protein, oneor more guide RNAs, a RNA-based masking construct; incubating the sampleor set of samples under conditions sufficient to allow binding of theone or more guide RNAs to one or more target molecules; activating theCRISPR effector protein via binding of the one or more guide RNAs to theone or more target molecules, wherein activating the CRISPR effectorprotein results in modification of the RNA-based masking construct suchthat a detectable positive signal is produced; and detecting thedetectable positive signal, wherein detection of the detectable positivesignal indicates a presence of one or more target molecules in thesample.

In another aspect, the invention provides a method for detectingpeptides in samples, comprising: distributing a sample or set of samplesinto a set of individual discrete volumes, the individual discretevolumes comprising peptide detection aptamers, a CRISPR systemcomprising an effector protein, one or more guide RNAs, a RNA-basedmasking construct, wherein the peptide detection aptamers comprising amasked RNA polymerase site and configured to bind one or more targetmolecules; incubating the sample or set of samples under conditionssufficient to allow binding of the peptide detection aptamers to the oneor more target molecules, wherein binding of the aptamer to acorresponding target molecule exposes the RNA polymerase binding siteresulting in RNA synthesis of a trigger RNA; activating the CRISPReffector protein via binding of the one or more guide RNAs to thetrigger RNA, wherein activating the CRISPR effector protein results inmodification of the RNA-based masking construct such that a detectablepositive signal is produced; and detecting the detectable positivesignal, wherein detection of the detectable positive signal indicates apresence of one or more target molecules in a sample.

In certain example embodiments, such methods further comprise amplifyingthe sample RNA or the trigger RNA. In other embodiments, amplifying RNAcomprises amplification by NASBA or RPA.

The low cost and adaptability of the assay platform lends itself to anumber of applications including (i) general RNA/DNA/proteinquantitation, (ii) rapid, multiplexed RNA/DNA and protein expressiondetection, and (iii) sensitive detection of target nucleic acids,peptides, and proteins in both clinical and environmental samples.Additionally, the systems disclosed herein may be adapted for detectionof transcripts within biological settings, such as cells. Given thehighly specific nature of the CRISPR effectors described herein, it maypossible to track allelic specific expression of transcripts ordisease-associated mutations in live cells.

In certain example embodiments, a single guide RNA specific to a singletarget is placed in separate volumes. Each volume may then receive adifferent sample or aliquot of the same sample. In certain exampleembodiments, multiple guide RNA each to separate target may be placed ina single well such that multiple targets may be screened in a differentwell. In order to detect multiple guide RNAs in a single volume, incertain example embodiments, multiple effector proteins with differentspecificities may be used. For example, different orthologs withdifferent sequence specificities may be used. For example, oneorthologue may preferentially cut A, while others preferentially cut C,U, or T. Accordingly, guide RNAs that are all, or comprise a substantialportion, of a single nucleotide may be generated, each with a differentfluorophore. In this way up to four different targets may be screened ina single individual discrete volume.

In certain aspects, the present invention relates to personalizedmedicine. In particular, the provision of a companion diagnostic tool asdescribed herein allows to tailor therapy, such as to overcome,circumvent, or prevent viral resistance, or to specifically targetparticular viral species, or strains which are prevalent in certainconditions, such as associated with particular geographic regions orparticular viral outbreaks (or stages of the outbreak).

It will be understood that the CRISPR/Cas system based detection methodsdescribed herein are non-limiting examples of suitabledetection/diagnostic systems.

In certain example embodiments, the systems, devices, and methods,disclosed herein are directed to detecting the presence of one or moreviral agents in a sample, such as a biological sample obtained from asubject. Accordingly, the methods disclosed herein can be adapted foruse in other methods (or in combination) with other methods that requirequick identification of microbe species, monitoring the presence ofmicrobial proteins (antigens), antibodies, antibody genes, detection ofcertain phenotypes (e.g. bacterial resistance), monitoring of diseaseprogression and/or outbreak, and antibiotic screening. Because of therapid and sensitive diagnostic capabilities of the embodiments disclosedhere, detection of microbe species type, down to a single nucleotidedifference, and the ability to be deployed as a POC device, theembodiments disclosed herein may be used guide therapeutic regimens,such as selection of the appropriate antibiotic or antiviral. Theembodiments disclosed herein may also be used to screen environmentalsamples (air, water, surfaces, food etc.) for the presence of microbialcontamination.

Particular embodiments disclosed herein describe methods and systemsthat will identify and distinguish viral species within a single sample,or across multiple samples, allowing for recognition of many differentviruses. The present methods allow the detection of pathogens anddistinguishing between two or more species of one or more virus or acombination thereof, in a biological or environmental sample, bydetecting the presence of a target nucleic acid sequence in the sample.A positive signal obtained from the sample indicates the presence of thevirus. Multiple viruses can be identified simultaneously using themethods and systems of the invention, by employing the use of more thanone effector protein, wherein each effector protein targets a specificmicrobial target sequence. In this way, a multi-level analysis can beperformed for a particular subject in which any number of microbes canbe detected at once. In some embodiments, simultaneous detection ofmultiple viruses may be performed using a set of probes that canidentify one or more viral species.

Multiplex analysis of samples enables large-scale detection of samples,reducing the time and cost of analyses. However, multiplex analyses areoften limited by the availability of a biological sample. In accordancewith the invention, however, alternatives to multiplex analysis may beperformed such that multiple effector proteins can be added to a singlesample and each masking construct may be combined with a separatequencher dye. In this case, positive signals may be obtained from eachquencher dye separately for multiple detection in a single sample.

Disclosed herein are methods for distinguishing between two or morespecies of one or more virus in a sample. The methods are also amenableto detecting one or more species of one or more virus in a sample.

Viral Detection

In some embodiments, a method for detecting viruses in samples isprovided comprising distributing a sample or set of samples into one ormore individual discrete volumes, the individual discrete volumescomprising a CRISPR system as described herein; incubating the sample orset of samples under conditions sufficient to allow binding of the oneor more guide RNAs to one or more microbe-specific targets; activatingthe CRISPR effector protein via binding of the one or more guide RNAs tothe one or more target molecules, wherein activating the CRISPR effectorprotein results in modification of the RNA-based masking construct suchthat a detectable positive signal is generated; and detecting thedetectable positive signal, wherein detection of the detectable positivesignal indicates a presence of one or more target molecules in thesample. The one or more target molecules may be mRNA, gDNA (coding ornon-coding), trRNA, or RNA comprising a target nucleotide tide sequencethat may be used to distinguish two or more microbial species/strainsfrom one another. The guide RNAs may be designed to detect targetsequences. The embodiments disclosed herein may also utilize certainsteps to improve hybridization between guide RNA and target RNAsequences. Methods for enhancing ribonucleic acid hybridization aredisclosed in WO 2015/085194, entitled “Enhanced Methods of RibonucleicAcid Hybridization” which is incorporated herein by reference. Themicrobe-specific target may be RNA or DNA or a protein. If DNA methodmay further comprise the use of DNA primers that introduce a RNApolymerase promoter as described herein. If the target is a protein thanthe method will utilized aptamers and steps specific to proteindetection described herein.

Detection of Single Nucleotide Variants

In some embodiments, one or more identified target sequences may bedetected using as a non-limiting example guide RNAs that are specificfor and bind to the target sequence as described herein. The systems andmethods of the present invention can distinguish even between singlenucleotide polymorphisms present among different viral species andtherefore, use of multiple guide RNAs in accordance with the inventionmay further expand on or improve the number of target sequences that maybe used to distinguish between species. For example, in someembodiments, the one or more guide RNAs may distinguish between virusesat the species, genus, family, order, class, phylum, kingdom, orphenotype, or a combination thereof.

Screening for Drug Resistance

In certain example embodiments, the devices, systems and methodsdisclosed herein may be used to screen for viral genes of interest, forexample antibiotic and/or antiviral resistance genes. As a non-limitingexample, guide RNAs may be designed to distinguish between known genesof interest. Samples, including clinical samples, may then be screenedusing the embodiments disclosed herein for detection of such genes. Theability to screen for drug resistance at POC would have tremendousbenefit in selecting an appropriate treatment regime.

Ribavirin is an effective antiviral that hits a number of RNA viruses.Several clinically important virues have evolved ribavirin resitanceincluding Foot and Mouth Disease Virus doi:10.1128/JVI.03594-13; poliovirus (Pfeifer and Kirkegaard. PNAS, 100(12):7289-7294, 2003); andhepatitis C virus (Pfeiffer and Kirkegaard, J. Virol. 79(4):2346-2355,2005). A number of other persistant RNA viruses, such as hepatitis andHIV, have evolved resitance to existing antiviral drugs: hepatitis Bvirus (lamivudine, tenofovir, entecavir) doi:10/1002/hep22900; hepatitsC virus (telaprevir, BILN2061, ITMN-191, SCh6, boceprevir, AG-021541,ACH-806) doi:10.1002/hep.22549; and HIV (many drug resistance mutations)hivb.standford.edu. The embodiments disclosed herein may be used todetect such variants among others.

Aside from drug resistance, there are a number of clinically relevantmutations that could be detected with the embodiments disclosed herein,such as persistent versus acute infection in LCMV(doi:10.1073/pnas.1019304108), and increased infectivity of Ebola (Diehlet al. Cell. 2016, 167(4):1088-1098.

As described herein elsewhere, closely related viral species (e.g.having only a single nucleotide difference in a given target sequence)may be distinguished by introduction of a synthetic mismatch in thegRNA, as described herein elsewhere.

Set Cover Approaches

In particular embodiments, all viral species within a defined set ofviruses can be identified.

The ability to detect multiple transcript abundances may allow for thegeneration of unique viral signatures indicative of a particularphenotype. Various machine learning techniques may be used to derive thegene signatures. Accordingly, for instance the guide RNAs of the CRISPRsystems may be used to identify and/or quantitate relative levels ofbiomarkers defined by the gene signature in order to detect certainphenotypes. In certain example embodiments, the gene signature indicatessusceptibility to an antibiotic, resistance to an antibiotic, or acombination thereof.

In one aspect of the invention, a method comprises detecting one or morevirus. In this manner, differentiation between infection of a subject byindividual viruses may be obtained. In some embodiments, suchdifferentiation may enable detection or diagnosis by a clinician ofspecific diseases, for example, different variants of a disease.Preferably the viral sequence is a genome of the pathogen or a fragmentthereof. The method further may comprise determining the evolution ofthe virus. Determining the evolution of the virus may compriseidentification of viral mutations, e.g. nucleotide deletion, nucleotideinsertion, nucleotide substitution. Amongst the latter, there arenon-synonymous, synonymous, and noncoding substitutions. Mutations aremore frequently non-synonymous during an outbreak. The method mayfurther comprise determining the substitution rate between two viralsequences analyzed as described above. Whether the mutations aredeleterious or even adaptive would require functional analysis, however,the rate of non-synonymous mutations suggests that continued progressionof this epidemic could afford an opportunity for pathogen adaptation,underscoring the need for rapid containment. Thus, the method mayfurther comprise assessing the risk of viral adaptation, wherein thenumber non-synonymous mutations is determined. (Gire, et al., Science345, 1369, 2014).

Monitoring Viral Outbreaks

In some embodiments, the methods as described herein may be used todetermine the evolution of a pathogen outbreak. The method may comprisedetecting one or more target sequences from a plurality of samples fromone or more subjects, wherein the target sequence is a sequence from amicrobe causing the outbreaks. Such a method may further comprisedetermining a pattern of pathogen transmission, or a mechanism involvedin a disease outbreak caused by a pathogen.

The pattern of pathogen transmission may comprise continued newtransmissions from the natural reservoir of the pathogen orsubject-to-subject transmissions (e.g. human-to-human transmission)following a single transmission from the natural reservoir or a mixtureof both. In one embodiment, the pathogen transmission may be bacterialor viral transmission, in such case, the target sequence is preferably amicrobial genome or fragments thereof. In one embodiment, the pattern ofthe pathogen transmission is the early pattern of the pathogentransmission, i.e. at the beginning of the pathogen outbreak.Determining the pattern of the pathogen transmission at the beginning ofthe outbreak increases likelihood of stopping the outbreak at theearliest possible time thereby reducing the possibility of local andinternational dissemination.

Determining the pattern of the pathogen transmission may comprisedetecting a pathogen sequence according to the methods described herein.Determining the pattern of the pathogen transmission may furthercomprise detecting shared intra-host variations of the pathogen sequencebetween the subjects and determining whether the shared intra-hostvariations show temporal patterns. Patterns in observed intrahost andinterhost variation provide important insight about transmission andepidemiology (Gire, et al., 2014).

Detection of shared intra-host variations between the subjects that showtemporal patterns is an indication of transmission links between subject(in particular between humans) because it can be explained by subjectinfection from multiple sources (superinfection), sample contaminationrecurring mutations (with or without balancing selection to reinforcemutations), or co-transmission of slightly divergent viruses that aroseby mutation earlier in the transmission chain (Park, et al., Cell161(7):1516-1526, 2015). Detection of shared intra-host variationsbetween subjects may comprise detection of intra-host variants locatedat common single nucleotide polymorphism (SNP) positions. Positivedetection of intra-host variants located at common (SNP) positions isindicative of superinfection and contamination as primary explanationsfor the intra-host variants. Superinfection and contamination can beparted on the basis of SNP frequency appearing as inter-host variants(Park, et al., 2015). Otherwise superinfection and contamination can beruled out. In this latter case, detection of shared intra-hostvariations between subjects may further comprise assessing thefrequencies of synonymous and nonsynonymous variants and comparing thefrequency of synonymous and nonsynonymous variants to one another. Anonsynonymous mutation is a mutation that alters the amino acid of theprotein, likely resulting in a biological change in the microbe that issubject to natural selection. Synonymous substitution does not alter anamino acid sequence. Equal frequency of synonymous and nonsynonymousvariants is indicative of the intra-host variants evolving neutrally. Iffrequencies of synonymous and nonsynonymous variants are divergent, theintra-host variants are likely to be maintained by balancing selection.If frequencies of synonymous and nonsynonymous variants are low, this isindicative of recurrent mutation. If frequencies of synonymous andnonsynonymous variants are high, this is indicative of co-transmission(Park, et al., 2015).

Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever withhigh case fatality rates. Andersen et al. generated a genomic catalog ofalmost 200 LASV sequences from clinical and rodent reservoir samples(Andersen, et al., Cell Volume 162, Issue 4, p 738-750, 13 Aug. 2015).Andersen et al. show that whereas the 2013-2015 EVD epidemic is fueledby human-to-human transmissions, LASV infections mainly result fromreservoir-to-human infections. Andersen et al. elucidated the spread ofLASV across West Africa and show that this migration was accompanied bychanges in LASV genome abundance, fatality rates, codon adaptation, andtranslational efficiency. The method may further comprisephylogenetically comparing a first pathogen sequence to a secondpathogen sequence, and determining whether there is a phylogenetic linkbetween the first and second pathogen sequences. The second pathogensequence may be an earlier reference sequence. If there is aphylogenetic link, the method may further comprise rooting the phylogenyof the first pathogen sequence to the second pathogen sequence. Thus, itis possible to construct the lineage of the first pathogen sequence.(Park, et al., 2015).

The method may further comprise determining whether the mutations aredeleterious or adaptive. Deleterious mutations are indicative oftransmission-impaired viruses and dead-end infections, thus normallyonly present in an individual subject. Mutations unique to oneindividual subject are those that occur on the external branches of thephylogenetic tree, whereas internal branch mutations are those presentin multiple samples (i.e. in multiple subjects). Higher rate ofnonsynonymous substitution is a characteristic of external branches ofthe phylogenetic tree (Park, et al., 2015).

In internal branches of the phylogenetic tree, selection has had moreopportunity to filter out deleterious mutants. Internal branches, bydefinition, have produced multiple descendent lineages and are thus lesslikely to include mutations with fitness costs. Thus, lower rate ofnonsynonymous substitution is indicative of internal branches (Park, etal., 2015).

Synonymous mutations, which likely have less impact on fitness, occurredat more comparable frequencies on internal and external branches (Park,et al., 2015).

By analyzing the sequenced target sequence, such as viral genomes, it ispossible to discover the mechanisms responsible for the severity of theepidemic episode such as during the 2014 Ebola outbreak. For example,Gire et al. made a phylogenetic comparison of the genomes of the 2014outbreak to all 20 genomes from earlier outbreaks suggests that the 2014West African virus likely spread from central Africa within the pastdecade. Rooting the phylogeny using divergence from other ebolavirusgenomes was problematic (6, 13). However, rooting the tree on the oldestoutbreak revealed a strong correlation between sample date androot-to-tip distance, with a substitution rate of 8×10-4 per site peryear (13). This suggests that the lineages of the three most recentoutbreaks all diverged from a common ancestor at roughly the same time,around 2004, which supports the hypothesis that each outbreak representsan independent zoonotic event from the same genetically diverse viralpopulation in its natural reservoir. They also found out that the 2014EBOV outbreak might be caused by a single transmission from the naturalreservoir, followed by human-to-human transmission during the outbreak.Their results also suggested that the epidemic episode in Sierra Leonmight stem from the introduction of two genetically distinct virusesfrom Guinea around the same time (Gire, et al., 2014).

It has been also possible to determine how the Lassa virus spread outfrom its origin point, in particular thanks to human-to-humantransmission and even retrace the history of this spread 400 years back(Andersen, et al., Cell 162(4):738-50, 2015).

In relation to the work needed during the 2013-2015 EBOV outbreak andthe difficulties encountered by the medical staff at the site of theoutbreak, and more generally, the method of the invention makes itpossible to carry out sequencing using fewer selected probes such thatsequencing can be accelerated, thus shortening the time needed fromsample taking to results procurement. Further, kits and systems can bedesigned to be usable on the field so that diagnostics of a patient canbe readily performed without need to send or ship samples to anotherpart of the country or the world.

In any method described above, sequencing the target sequence orfragment thereof may used any of the sequencing processes describedabove. Further, sequencing the target sequence or fragment thereof maybe a near-real-time sequencing. Sequencing the target sequence orfragment thereof may be carried out according to previously describedmethods (Experimental Procedures: Matranga et al., 2014; and Gire, etal., 2014). Sequencing the target sequence or fragment thereof maycomprise parallel sequencing of a plurality of target sequences.Sequencing the target sequence or fragment thereof may comprise Illuminasequencing.

Analyzing the target sequence or fragment thereof that hybridizes to oneor more of the selected probes may be an identifying analysis, whereinhybridization of a selected probe to the target sequence or a fragmentthereof indicates the presence of the target sequence within the sample.

Currently, primary diagnostics are based on the symptoms a patient has.However, various diseases may share identical symptoms so thatdiagnostics rely much on statistics. For example, malaria triggersflu-like symptoms: headache, fever, shivering, joint pain, vomiting,hemolytic anemia, jaundice, hemoglobin in the urine, retinal damage, andconvulsions. These symptoms are also common for septicemia,gastroenteritis, and viral diseases. Amongst the latter, Ebolahemorrhagic fever has the following symptoms fever, sore throat,muscular pain, headaches, vomiting, diarrhea, rash, decreased functionof the liver and kidneys, internal and external hemorrhage.

When a patient is presented to a medical unit, for example in tropicalAfrica, basic diagnostics will conclude to malaria becausestatistically, malaria is the most probable disease within that regionof Africa. The patient is consequently treated for malaria although thepatient might not actually have contracted the disease and the patientends up not being correctly treated. This lack of correct treatment canbe life-threatening especially when the disease the patient contractedpresents a rapid evolution. It might be too late before the medicalstaff realizes that the treatment given to the patient is ineffectiveand comes to the correct diagnostics and administers the adequatetreatment to the patient.

The method of the invention provides a solution to this situation.Indeed, because the number of guide RNAs can be dramatically reduced,this makes it possible to provide on a single chip selected probesdivided into groups, each group being specific to one disease, such thata plurality of diseases, e.g. viral infection, can be diagnosed at thesame time. Thanks to the invention, more than 3 diseases can bediagnosed on a single chip, preferably more than 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 diseases at the same time,preferably the diseases that most commonly occur within the populationof a given geographical area. Since each group of selected probes isspecific to one of the diagnosed diseases, a more accurate diagnosticscan be performed, thus diminishing the risk of administering the wrongtreatment to the patient.

In other cases, a disease such as a viral infection may occur withoutany symptoms, or had caused symptoms but they faded out before thepatient is presented to the medical staff. In such cases, either thepatient does not seek any medical assistance or the diagnostics iscomplicated due to the absence of symptoms on the day of thepresentation.

The present invention may also be used in concert with other methods ofdiagnosing disease, identifying pathogens and optimizing treatment basedupon detection of nucleic acids, such as mRNA in crude, non-purifiedsamples (see, e.g., US patent publication No.).

The method of the invention also provides a powerful tool to addressthis situation. Indeed, since a plurality of groups of selected guideRNAs, each group being specific to one of the most common diseases thatoccur within the population of the given area, are comprised within asingle diagnostic, the medical staff only need to contact a biologicalsample taken from the patient with the chip. Reading the chip revealsthe diseases the patient has contracted.

In some cases, the patient is presented to the medical staff fordiagnostics of particular symptoms. The method of the invention makes itpossible not only to identify which disease causes these symptoms but atthe same time determine whether the patient suffers from another diseasehe was not aware of.

This information might be of utmost importance when searching for themechanisms of an outbreak. Indeed, groups of patients with identicalviruses also show temporal patterns suggesting a subject-to-subjecttransmission links.

Sample Types

Appropriate samples for use in the methods disclosed herein include anyconventional biological sample obtained from an organism or a partthereof, such as a plant, animal, bacteria, and the like. In particularembodiments, the biological sample is obtained from an animal subject,such as a human subject. A biological sample is any solid or fluidsample obtained from, excreted by or secreted by any living organism,including, without limitation, single celled organisms, such asbacteria, yeast, protozoans, and amoebas among others, multicellularorganisms (such as plants or animals, including samples from a healthyor apparently healthy human subject or a human patient affected by acondition or disease to be diagnosed or investigated, such as aninfection with a pathogenic microorganism, such as a pathogenic bacteriaor virus). For example, a biological sample can be a biological fluidobtained from, for example, blood, plasma, serum, urine, stool, sputum,mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion,seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or anybodily secretion, a transudate, an exudate (for example, fluid obtainedfrom an abscess or any other site of infection or inflammation), orfluid obtained from a joint (for example, a normal joint or a jointaffected by disease, such as rheumatoid arthritis, osteoarthritis, goutor septic arthritis), or a swab of skin or mucosal membrane surface.

A sample can also be a sample obtained from any organ or tissue(including a biopsy or autopsy specimen, such as a tumor biopsy) or caninclude a cell (whether a primary cell or cultured cell) or mediumconditioned by any cell, tissue or organ. Exemplary samples include,without limitation, cells, cell lysates, blood smears, cytocentrifugepreparations, cytology smears, bodily fluids (e.g., blood, plasma,serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.),tissue biopsies (e.g., tumor biopsies), fine-needle aspirates, and/ortissue sections (e.g., cryostat tissue sections and/or paraffin-embeddedtissue sections). In other examples, the sample includes circulatingtumor cells (which can be identified by cell surface markers). Inparticular examples, samples are used directly (e.g., fresh or frozen),or can be manipulated prior to use, for example, by fixation (e.g.,using formalin) and/or embedding in wax (such as formalin-fixedparaffin-embedded (FFPE) tissue samples). It will appreciated that anymethod of obtaining tissue from a subject can be utilized, and that theselection of the method used will depend upon various factors such asthe type of tissue, age of the subject, or procedures available to thepractitioner. Standard techniques for acquisition of such samples areavailable in the art. See, for example Schluger et al., J Exp. Med.176:1327-33 (1992); Bigby et al., Am. Rev. Respir. Dis. 133:515-18(1986); Kovacs et al., NEJM 318:589-93 (1988); and Ognibene et al., Am.Rev. Respir. Dis. 129:929-32 (1984).

In other embodiments, a sample may be an environmental sample, such aswater, soil, or a surface such as industrial or medical surface. In someembodiments, methods such as disclosed in US patent publication No.2013/0190196 may be applied for detection of nucleic acid signatures,specifically RNA levels, directly from crude cellular samples with ahigh degree of sensitivity and specificity. Sequences specific to eachpathogen of interest may be identified or selected by comparing thecoding sequences from the pathogen of interest to all coding sequencesin other organisms by BLAST software.

Several embodiments of the present disclosure involve the use ofprocedures and approaches known in the art to successfully fractionateclinical blood samples. See, e.g. the procedure described in Han Wei Houet al., Microfluidic Devices for Blood Fractionation, Micromachines2011, 2, 319-343; Ali Asgar S. Bhagat et al., Dean Flow Fractionation(DFF) Isolation of Circulating Tumor Cells (CTCs) from Blood, 15^(th)International Conference on Miniaturized Systems for Chemistry and LifeSciences, Oct. 2-6, 2011, Seattle, Wash.; and International PatentPublication No. WO2011109762, the disclosures of which are hereinincorporated by reference in their entirety. Blood samples are commonlyexpanded in culture to increase sample size for testing purposes. Insome embodiments of the present invention, blood or other biologicalsamples may be used in methods as described herein without the need forexpansion in culture.

Further, several embodiments of the present disclosure involve the useof procedures and approaches known in the art to successfully isolatepathogens from whole blood using spiral microchannel, as described inHan Wei Hou et al., Pathogen Isolation from Whole Blood Using SpiralMicrochannel, Case No. 15995JR, Massachusetts Institute of Technology,manuscript in preparation, the disclosure of which is hereinincorporated by reference in its entirety.

Owing to the increased sensitivity of the embodiments disclosed herein,in certain example embodiments, the assays and methods may be run oncrude samples or samples where the target molecules to be detected arenot further fractionated or purified from the sample.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1. C2c2 Prevents Infection and Reduces Replication ofLymphocytic Choriomeningitis Virus (LCMV)

Guide RNAs were designed to bind to various areas of the LCMV genome, asindicated in FIGS. 1A and 1B, and are listed in Table 12 below.

TABLE 12 gRNA SEQ ID annotation gRNA sequence NO L1ctcacgaagaaagttgtgcaaccaaaca 173 L2 tcttcaatctggttggtaatggatattt 174 L3gccaatttgttagtgtcctctataaatt 175 L4 tatctcacagaccctatttgattttgcc 176 L5aaattettcattaaattcaccatttttg 177 L6 tatagtttaaacataactctctcaattc 178 S1atccaaaaagcctaggatccccggtgcg 179 S2 agaatgtcaagttgtattggatggttat 180 S3aaagcagccttgttgtagtcaattagtc 181 S4 tgatctctttcttctttttgtcccttac 182 S5tctctttcttctttttgtcccttactat 183 S6 caatcaaatgcctaggatccactgtgcg 184

A plasmid expressing Leptotrichia wadei (Lw) C2c2/Cas13a fused to msfGFPwith a nuclear export signal, and a plasmid expressing guide RNA weretransfected into 293FT cells using Lipofectamine 2000. 293FT cells wereplated simultaneously to lipofectamine-plasmid mixture addition. After24 h, the transfected 293FT cells were infected with LCMV armstrong (MOI5, 1 h) (viral titer was determined by focus forming unit assay withVero cells). After 1 hour of infection, cells were washed with citratebuffer to destroy virus remaining in the infection media that did notinfect the cells. 48 hours post infection, virus-containing supernatantwas removed and diluted 1:10 in Nuclease-free water and then used asinput for RT-qPCR with primers against LCMV GP. The results are shown inFIG. 2B.

293FT cells were plated 1 day prior to LCMV infection, so that infectionwas performed at approximately 80-85% confluency. 24 h post plating,293FT cells were infected with LCMV Armstrong (MOI 5, 1 h) (viral titerwas determined by focus forming unit assay with Vero cells). After 1hour of infection, cells were washed with citrate buffer to destroyvirus remaining in the infection media that did not infect the cells.After 24 h, a plasmid expressing Lw C2c2/Cas13a fused to msfGFP with anuclear export signal, and a plasmid expressing guide RNA wastransfected into 293FT cells using Lipofectamine 2000. 48 h afterinfection, virus-containing supernatant was removed and diluted 1:10 inNuclease-free water and then used as input for RT-qPCR with primersagainst LCMV GP. The results are shown in FIG. 3.

A plasmid expressing Lw C2c2/Cas13a fused to msfGFP with a nuclearexport signal, and either a single plasmid or multiple plasmidsexpressing guide RNA(s) were transfected into 293FT cells usingLipofectamine 2000. 293FT cells were plated simultaneously tolipofectamine-plasmid mixture addition. Cells were transfected with 1 to4 different guide RNAs. After 24 h, the cells were infected with LCMVArmstrong (MOI 5, 1 h) (viral titer was determined by focus forming unitassay with Vero cells). After 1 hour of infection, cells were washedwith citrate buffer to destroy virus remaining in the infection mediathat did not infect the cells. 48 hours post infection, virus-containingsupernatant was removed and diluted 1:10 in Nuclease-free water and thenused as input for RT-qPCR with primers against LCMV GP. The results areshown in FIG. 4.

From FIGS. 2B-4, it is clear that the CRISPR system designed to targetviral RNA can reduce viral replication and thus viral load, which iseven more pronounced when using multiple guide RNAs.

Example 2. Whole Genome Screen of LCMV for Cas13 Targeting Guides

A genome-wide screen was performed to identify the most efficient guideRNAs. 283 guide RNAs are tiled across the LCMV genome: every 50 nt onthe coding strand (207 guides) and every 150 nt on the non-coding strand(76 guides).

LCMV Screen design and approach: To conduct a full-genome screen forguides that reduce LCMV replication guides were tiled across both the Sand L segment of the LCMV genome. For coding regions, a 28 nt guide wasdesigned every 50 nt along the coding region. For non-coding regions,guides were designed every 150 nt. There are 4 coding regions for LCMVfor the four proteins (GPC, NP (or dN), Z and L) and each of these 4proteins has their own non-coding region. With this strategy, 283 guideswere tested total: GPC: 11 guides in the non-coding region and 32 guidesin the coding region; NP: 12 guides in the non-coding region and 35guides in the coding region; Z: 4 guides in the non-coding region and 7guides in the coding region; L: 49 guides in the non-coding region and133 guides in the coding region. The spacer sequences of the respectivegRNAs, as well as their relative positions within the LCMV genome andLMCV genes are provided in Table 13 below.

TABLE 13 ID Spacer sequence SEQ ID No.   1 L_targ_Z_Lsh/Lw2/Ca/LbFSL_1gttactcttcgtagggaggtggagagct   1 185   2 L_targ_Z_Lsh/Lw2/Ca/LbFSL_2ctggttggtaatggatatttacaaagag   2 186   3 L_targ_Z_Lsh/Lw2/Ca/LbFSL_3cagaaggtttaaacagtgcctgcaaagg   3 187   4 L_targ_Z_Lsh/Lw2/Ca/LbFSL_4agctgtcaaatttctgccagcaagattt   4 188   5 L_targ_Z_Lsh/Lw2/Ca/LbFSL_5gtggtatctggtaggatttcggccctgt   5 189   6 L_targ_Z_Lsh/Lw2/Ca/LbFSL_6ctctctggacttgccttgacccatcgct   6 190   7 L_targ_Z_Lsh/Lw2/Ca/LbFSL_7agcctcacgaagaaagttgtgcaaccaa   7 191 (START OF L)   1L_targ_L_Lsh/Lw2/Ca/LbFSL_1 tcagtcgatgtcctcggccaccgacccg   8 192   2L_targ_L_Lsh/Lw2/Ca/LbFSL_2 tcccottgagtctaaacctgccccccac   9 193   3L_targ_L_Lsh/Lw2/Ca/LbFSL_3 ctagatttgctaaaacaaagtctgcaat  10 194   4L_targ_L_Lsh/Lw2/Ca/LbFSL_4 caaaagcgacagtggaatcagcagaata  11 195   5L_targ_L_Lsh/Lw2/Ca/LbFSL_5 ggaggattacacttatctctgaacccaa  12 196   6L_targ_L_Lsh/Lw2/Ca/LbFSL_6 cgatgcaggaagaggttcccaaggacat  13 197   7L_targ_L_Lsh/Lw2/Ca/LbFSL_7 aagtcctgctagaaagactttcatgtcc  14 198   8L_targ_L_Lsh/Lw2/Ca/LbFSL_8 tattttggacaaggtttcttccttcaaa  15 199   9L_targ_L_Lsh/Lw2/Ca/LbFSL_9 agtggcacaggctcccactcaggtccaa  16 200  10L_targ_L_Lsh/Lw2/Ca/LbFSL_10 aatcccatccagtattcttttggagccc  17 201  11L_targ_L_Lsh/Lw2/Ca/LbFSL_11 caccaagtatcaagggatcttccatgta  18 202  12L_targ_L_Lsh/Lw2/Ca/LbFSL_12 atatcaaagacaccatcgttcaccttga  19 203  13L_targ_L_Lsh/Lw2/Ca/LbFSL_13 gtggaggcattcatccaacattcttcta  20 204  14L_targ_L_Lsh/Lw2/Ca/LbFSL_14 gagagcatgataaaagttcagccacacc  21 205  15L_targ_L_Lsh/Lw2/Ca/LbFSL_15 accaagaatatcaatgaaaatttcctta  22 206  16L_targ_L_Lsh/Lw2/Ca/LbFSL_16 gtgcgtaaagtccactgaaattgaaaac  23 207  17L_targ_L_Lsh/Lw2/Ca/LbFSL_17 tgagcatgtagtcccacagatcctttaa  24 208  18L_targ_L_Lsh/Lw2/Ca/LbFSL_18 gtcaggccctgcctaatcaacatggcag  25 209  19L_targ_L_Lsh/Lw2/Ca/LbFSL_19 tcggtaagagaaccacccaaaaccaaac  26 210  20L_targ_L_Lsh/Lw2/Ca/LbFSL_20 gcctctccacatttttgttcaccacctt  27 211  21L_targ_L_Lsh/Lw2/Ca/LbFSL_21 cccagtgcctcagcaccatcttcagatg  28 212  22L_targ_L_Lsh/Lw2/Ca/LbFSL_22 ccatgaaaaattgcctaatgtcctggtt  29 213  23L_targ_L_Lsh/Lw2/Ca/LbFSL_23 atgattcaaaatacacctgttttaagaa  30 214  24L_targ_L_Lsh/Lw2/Ca/LbFSL_24 ctaacaacaaattcatcaaccagactgg  31 215  25L_targ_L_Lsh/Lw2/Ca/LbFSL_25 ggcaaggtcagaaaacagaacagtgtaa  32 216  26L_targ_L_Lsh/Lw2/Ca/LbFSL_26 caacatgagaaatgagtgacaaggattc  33 217  27L_targ_L_Lsh/Lw2/Ca/LbFSL_27 cagaggtcaaggaatttaattctgggac  34 218  28L_targ_L_Lsh/Lw2/Ca/LbFSL_28 catgtcagacataaatggaagaagctga  35 219  29L_targ_L_Lsh/Lw2/Ca/LbFSL_29 accgcctcacagattgaatcacttggtt  36 220  30L_targ_L_Lsh/Lw2/Ca/LbFSL_30 agccttgagctctcaggctttcttgcta  37 221  31L_targ_L_Lsh/Lw2/Ca/LbFSL_31 cttaagagttaggttctcactgttattc  38 222  32L_targ_L_Lsh/Lw2/Ca/LbFSL_32 ggacccaaacacccaactcaaaagagtt  39 223  33L_targ_L_Lsh/Lw2/Ca/LbFSL_33 tcccaaagaagaggccttaaaaggcata  40 224  34L_targ_L_Lsh/Lw2/Ca/LbFSL_34 atgagactgtttgtcacaaatgtacagc  41 225  35L_targ_L_Lsh/Lw2/Ca/LbFSL_35 ctcttgtcacatgatcatctgtggttag  42 226  36L_targ_L_Lsh/Lw2/Ca/LbFSL_36 tacagattttccctattttigtttctca  43 227  37L_targ_L_Lsh/Lw2/Ca/LbFSL_37 gcaaaggcctataaagccagatgagata  44 228  38L_targ_L_Lsh/Lw2/Ca/LbFSL_38 tgattgcttctgacagcagcttctgtgc  45 229  39L_targ_L_Lsh/Lw2/Ca/LbFSL_39 agtttgttctggagtgtcttgatcaatg  46 230  40L_targ_L_Lsh/Lw2/Ca/LbFSL_40 agtcatcactgatggataaaccaccttt  47 231  41L_targ_L_Lsh/Lw2/Ca/LbFSL_41 ggaacatttcattcaaattcaaccagtt  48 232  42L_targ_L_Lsh/Lw2/Ca/LbFSL_42 tcttcttcaagaccgaggaggtctccca  49 233  43L_targ_L_Lsh/Lw2/Ca/LbFSL_43 atctctgttaaataggtctaagaaaaat  50 234  44L_targ_L_Lsh/Lw2/Ca/LbFSL_44 tgagcttatgatgcagtttccttacaag  51 235  45L_targ_L_Lsh/Lw2/Ca/LbFSL_45 ttaggacacagttcctcaatgagtcttt  52 236  46L_targ_L_Lsh/Lw2/Ca/LbFSL_46 atccagccaatctttcacatcagtgttg  53 237  47L_targ_L_Lsh/Lw2/Ca/LbFSL_47 aagggaaattggcatactttaggaggtc  54 238  48L_targ_L_Lsh/Lw2/Ca/LbFSL_48 ttaactagggagactgggacgccatttg  55 239  49L_targ_L_Lsh/Lw2/Ca/LbFSL_49 atctattgtttcacaaagttgatgtggc  56 240  50L_targ_L_Lsh/Lw2/Ca/LbFSL_50 gcgctgcagatacaaactttgtgagaag  57 241  51L_targ_L_Lsh/Lw2/Ca/LbFSL_51 tagaatctagatttaaattctgcagcga  58 242  52L_targ_L_Lsh/Lw2/Ca/LbFSL_52 gctgataaatttgtttaacaagccgctc  59 243  53L_targ_L_Lsh/Lw2/Ca/LbFSL_53 ggacaaggacttcctccggatcacttac  60 244  54L_targ_L_Lsh/Lw2/Ca/LbFSL_54 tcaaataaagtgatctgatcatcacttg  61 245  55L_targ_L_Lsh/Lw2/Ca/LbFSL_55 gccaaagataacaccaatgcagtagttg  62 246  56L_targ_L_Lsh/Lw2/Ca/LbFSL_56 catagaagtcagaagcattatgcaagat  63 247  57L_targ_L_Lsh/Lw2/Ca/LbFSL_57 ctggatatatgggatggcactatcccca  64 248  58L_targ_L_Lsh/Lw2/Ca/LbFSL_58 tctctcagtaacagttgtttctgaaccc  65 249  59L_targ_L_Lsh/Lw2/Ca/LbFSL_59 tgacatatgatttcatcattgcattcac  66 250  60L_targ_L_Lsh/Lw2/Ca/LbFSL_60 agcttatgcatgtgccaagttaacaaag  67 251  61L_targ_L_Lsh/Lw2/Ca/LbFSL_61 acgcacatactggtcatcacctagtttg  68 252  62L_targ_L_Lsh/Lw2/Ca/LbFSL_62 acaaaaatgggcacatcattggtcccca  69 253  63L_targ_L_Lsh/Lw2/Ca/LbFSL_63 tttaagaacccttcccgcacattgatag  70 254  64L_targ_L_Lsh/Lw2/Ca/LbFSL_64 aaattccttatcattgtttaaacaggag  71 255  65L_targ_L_Lsh/Lw2/Ca/LbFSL_65 actcaaaataatcttctattaaccttgt  72 256  66L_targ_L_Lsh/Lw2/Ca/LbFSL_66 ccaatatagagttctctatttcccccaa  73 257  67L_targ_L_Lsh/Lw2/Ca/LbFSL_67 aaatttcagccttccagagtcaggacct  74 258  68L_targ_L_Lsh/Lw2/Ca/LbFSL_68 attcttctgagtagaagcacagattttt  75 259  69L_targ_L_Lsh/Lw2/Ca/LbFSL_69 gtcaacgacagagctttactaagggact  76 260  70L_targ_L_Lsh/Lw2/Ca/LbFSL_70 gattctcacgtcttcttccagtttgtcc  77 261  71L_targ_L_Lsh/Lw2/Ca/LbFSL_71 cttgcctttgcatatgcctgtatttccc  78 262  72L_targ_L_Lsh/Lw2/Ca/LbFSL_72 tgcaacagaatcatcttcatgcaagaaa  79 263  73L_targ_L_Lsh/Lw2/Ca/LbFSL_73 ctttctacaaaggttttttgccatctca  80 264  74L_targ_L_Lsh/Lw2/Ca/LbFSL_74 tgactgaggtgaaatacaaaggtgacag  81 265  75L_targ_L_Lsh/Lw2/Ca/LbFSL_75 tcacagataaatttcatgtcatcattgg  82 266  76L_targ_L_Lsh/Lw2/Ca/LbFSL_76 ttctactaaatggaaagatatttctgac  83 267  77L_targ_L_Lsh/Lw2/Ca/LbFSL_77 ccatcttccctgttagaataagctgtaa  84 268  78L_targ_L_Lsh/Lw2/Ca/LbFSL_78 gtaagtttttctccatctcctttgtcat  85 269  79L_targ_L_Lsh/Lw2/Ca/LbFSL_79 ccgtgctattgtggtgttgaccttttct  86 270  80L_targ_L_Lsh/Lw2/Ca/LbFSL_80 tctcttcttctccatcaaaacatatttc  87 271  81L_targ_L_Lsh/Lw2/Ca/LbFSL_81 cctgtctcttctcccttggaaccgatga  88 272  82L_targ_L_Lsh/Lw2/Ca/LbFSL_82 aactttatattcatagtctgagtggctc  89 273  83L_targ_L_Lsh/Lw2/Ca/LbFSL_83 cgaaactctccgtaatttgactcacagc  90 274  84L_targ_L_Lsh/Lw2/Ca/LbFSL_84 tcatattccagaagtcgttctccattta  91 275  85L_targ_L_Lsh/Lw2/Ca/LbFSL_85 tttgttactagcaagatctaatgctgtc  92 276  86L_targ_L_Lsh/Lw2/Ca/LbFSL_86 gatctaggctgtttagcttcttctctcc  93 277  87L_targ_L_Lsh/Lw2/Ca/LbFSL_87 ttaaatgaagacaccattaggctaaagg  94 278  88L_targ_L_Lsh/Lw2/Ca/LbFSL_88 tgtatgctgacagtcaatttctttacta  95 279  89L_targ_L_Lsh/Lw2/Ca/LbFSL_89 agaacacacattcttcctcaggagtgat  96 280  90L_targ_L_Lsh/Lw2/Ca/LbFSL_90 aaaccaaattgacttttgggctcaaaga  97 281  91L_targ_L_Lsh/Lw2/Ca/LbFSL_91 atctgttagcctgtcaggggtctccttt  98 282  92L_targ_L_Lsh/Lw2/Ca/LbFSL_92 acacattcaacataaatttaaattttgc  99 283  93L_targ_L_Lsh/Lw2/Ca/LbFSL_93 gtaccaaaaatagtttttattaggaatc 100 284  94L_targ_L_Lsh/Lw2/Ca/LbFSL_94 ctcagcaggtgtgatcagatcctccctc 101 285  95L_targ_L_Lsh/Lw2/Ca/LbFSL_95 atgagaaatctgacactattgccatcac 102 286  96L_targ_L_Lsh/Lw2/Ca/LbFSL_96 tgcttttgatttctctttgttgggttgg 103 287  97L_targ_L_Lsh/Lw2/Ca/LbFSL_97 cctcagtgcaacctcaatgtcggtgaga 104 288  98L_targ_L_Lsh/Lw2/Ca/LbFSL_98 atctaatccatgaaatcatgatgtctat 105 289  99L_targ_L_Lsh/Lw2/Ca/LbFSL_99 gaaaaaattggtaaaaagaaccttttag 106 290 100L_targ_L_Lsh/Lw2/Ca/LbFSL_100 atgaccatccgggccttgtatggagtag 107 291 101L_targ_L_Lsh/Lw2/Ca/LbFSL_101 tctggtataataggtggtattcttcaga 108 292 102L_targ_L_Lsh/Lw2/Ca/LbFSL_102 aacacttctttgcattctaccacttgat 109 293 103L_targ_L_Lsh/Lw2/Ca/LbFSL_103 ttgccttagtctagcaactgagctagtt 110 294 104L_targ_L_Lsh/Lw2/Ca/LbFSL_104 gacaaacagatgataatcttctcaggct 111 295 105L_targ_L_Lsh/Lw2/Ca/LbFSL_105 gtgctgggttggaaattgtaatcttcaa 112 296 106L_targ_L_Lsh/Lw2/Ca/LbFSL_106 gtgagctccaattttcataaagttctca 113 297 107L_targ_L_Lsh/Lw2/Ca/LbFSL_107 attcttgctcaaggtgttcagacagtcc 114 298 108L_targ_L_Lsh/Lw2/Ca/LbFSL_108 accactaacaggcatttttgaatttttg 115 299 109L_targ_L_Lsh/Lw2/Ca/LbFSL_109 cctaaacaattcctcaaaagacaccttt 116 300 110L_targ_L_Lsh/Lw2/Ca/LbFSL_110 tattcctcaaaagtctaatgaactcctc 117 301 111L_targ_L_Lsh/Lw2/Ca/LbFSL_111 agcctatcattcacactactatagcaac 118 302 112L_targ_L_Lsh/Lw2/Ca/LbFSL_112 ttttaaccctttgaatttcgactgtttt 119 303 113L_targ_L_Lsh/Lw2/Ca/LbFSL_113 catccagatttaacaactgtctccttct 120 304 114L_targ_L_Lsh/Lw2/Ca/LbFSL_114 ttgactttgtttaacatagagaggagcc 121 305 115L_targ_L_Lsh/Lw2/Ca/LbFSL_115 acttcccctttcgtgcccatgggtctct 122 306 116L_targ_L_Lsh/Lw2/Ca/LbFSL_116 gacaggattccactgcctccctgcttaa 123 307 117L_targ_L_Lsh/Lw2/Ca/LbFSL_117 gcaaggttttcatagagctcagagaatt 124 308 118L_targ_L_Lsh/Lw2/Ca/LbFSL_118 tactttctgaaagtttctctttaatttc 125 309 119L_targ_L_Lsh/Lw2/Ca/LbFSL_119 acctgctgaaaagagagtttattccaaa 126 310 120L_targ_L_Lsh/Lw2/Ca/LbFSL_120 ttggggttgatgccttcgtggcacatcc 127 311 121L_targ_L_Lsh/Lw2/Ca/LbFSL_121 tgacctcgcatctttcagaattttcata 128 312 122L_targ_L_Lsh/Lw2/Ca/LbFSL_122 cgatagtagtcttcagggactcacagag 129 313 123L_targ_L_Lsh/Lw2/Ca/LbFSL_123 aagactttctcattttggttagaatact 130 314 124L_targ_L_Lsh/Lw2/Ca/LbFSL_124 tctaaatttgaagtttgcccactctggc 131 315 125L_targ_L_Lsh/Lw2/Ca/LbFSL_125 aacgaccatctactattggaactaatgt 132 316 126L_targ_L_Lsh/Lw2/Ca/LbFSL_126 tccctgatgcatgccaatttgttagtgt 133 317 127L_targ_L_Lsh/Lw2/Ca/LbFSL_127 actggctggagtgctcctaacaaaacac 134 318 128L_targ_L_Lsh/Lw2/Ca/LbFSL_128 ctatcagcttgtaaccatcaggaatgat 135 319 129L_targ_L_Lsh/Lw2/Ca/LbFSL_129 attccagactccaccaaaattgtttcca 136 320 130L_targ_L_Lsh/Lw2/Ca/LbFSL_130 tgtgcagccactcttgtctgcactgtct 137 321 131L_targ_L_Lsh/Lw2/Ca/LbFSL_131 acttgagtccctcaatcagaaccattct 138 322 132L_targ_L_Lsh/Lw2/Ca/LbFSL_132 ttgagtttctgccttgacaacctctcat 139 323START OF GPC 133 L_targ_L_Lsh/Lw2/Ca/LbFSL_133taactctctcaattctgagatgatttca 140 324   1 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_1ttcagcgtcttttccagacggtttttac 141 325   2 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_2caactacaaattcctttgttggttaatc 142 326   3 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_3accttttatgtgcctgtgtgttggtatt 143 327   4 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_4tgactagatatgcagatgtggaaaacat 144 328   5 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_5ggggtactcccctgcctctttatgtaat 145 329   6 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_6catgttatcggcttcctgttcgatttga 146 330   7 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_7agtaagaaccattggtgacaagccagca 147 331   8 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_8gtctttgcatgttctaggtaccaaaact 148 332   9 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_9ccccatcagatctctcaagtggttcctc 149 333  10 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_10aagaattcactgttgttttgaataagtg 150 334  11 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_11ttgaacttactcaaagcagccttgttgt 151 335  12 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_12acagaattcttcatcatgatttacattg 152 336  13 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_13cgaaacacttaagctctgcagcaagaat 153 337  14 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_14ccacctggattctccacccctgaagagt 154 338  15 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_15gcccgctagtctcctagtgaagaactta 155 339  16 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_16tcctggacatcccaaaaggacctgcata 156 340  17 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_17ctatitigtataatcaggtattggtaac 157 341  18 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_18cttgccatctgagcctgtccagccccag 158 342  19 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_19cgaaggcagttctaaacatatctaggac 159 343  20 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_20tggctctgagcactttgtgcatttgaga 160 344  21 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_21tatgccattgttgaagtcgcaggatact 161 345  22 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_22tgatactgaggtgtaggctcgaaactat 162 346  23 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_23gtctttttattgaaggcagaggtcagat 163 347  24 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_24ggaatcattggtgaaggtcaattctagt 164 348  25 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_25aatggtgggagttgttggctgaacatgc 165 349  26 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_26tgtgacatatcaaactccactgacttaa 166 350  27 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_27gtcgggtcccttaagaccgtacatgcca 167 351  28 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_28ggaaactgatcaatgcgaatatcccaca 168 352  29 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_29ttgatacccgtgatcacgataagcacaa 169 353  30 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_30atcgatgatgtgaggcagagcctcaaac 170 354  31 S_targ_GPC_Lsh/Lw2/Ca/LbFSL_31ttctgtaggatagggcctgacacccagt 171 355 START OF NP  32S_targ_GPC_Lsh/Lw2/Ca/LbFSL_32 caaaaagcctaggatccccggtgcg 172 356   1S_targ_NP_Lsh/Lw2/Ca/LbFSL_1 cttagagtgtcacaacatttgggcctct 173 357   2S_targ_NP_Lsh/Lw2/Ca/LbFSL_2 atgttgtgaacagttttcagatctggga 174 358   3S_targ_NP_Lsh/Lw2/Ca/LbFSL_3 aaaaatgatgcagtccatgagtgcacag 175 359   4S_targ_NP_Lsh/Lw2/Ca/LbFSL_4 ttttgtcccttactattccagtatgcat 176 360   5S_targ_NP_Lsh/Lw2/Ca/LbFSL_5 tcccacactttgtcttcatactccctcg 177 361   6S_targ_NP_Lsh/Lw2/Ca/LbFSL_6 atcgataagcttaatgtccttcctattc 178 362   7S_targ_NP_Lsh/Lw2/Ca/LbFSL_7 tgtcatcggagccttgacagcttagaac 179 363   8S_targ_NP_Lsh/Lw2/Ca/LbFSL_8 ataactgacgaggtcaacccgggttgcg 180 364   9S_targ_NP_Lsh/Lw2/Ca/LbFSL_9 catgccgtgtgagtacttggaatcttgc 181 365  10S_targ_NP_Lsh/Lw2/Ca/LbFSL_10 gttccctgtaaaagtgtatgaactgccc 182 366  11S_targ_NP_Lsh/Lw2/Ca/LbFSL_11 atttccactggatcattaaatctaccct 183 367  12S_targ_NP_Lsh/Lw2/Ca/LbFSL_12 gttggggtcaattcctcccatgaggtct 184 368  13S_targ_NP_Lsh/Lw2/Ca/LbFSL_13 agcttaagcccacctgaggtggacctgc 185 369  14S_targ_NP_Lsh/Lw2/Ca/LbFSL_14 gagttgactgcaggtttctcgcttgtga 186 370  15S_targ_NP_Lsh/Lw2/Ca/LbFSL_15 tgctctccccacaatcgatgttctacaa 187 371  16S_targ_NP_Lsh/Lw2/Ca/LbFSL_16 ctgaaaggcaaactttatagaggatgtt 188 372  17S_targ_NP_Lsh/Lw2/Ca/LbFSL_17 acttggtctgaaacaaacatgttgagtt 189 373  18S_targ_NP_Lsh/Lw2/Ca/LbFSL_18 cttcaagaggtcctcgctgttgcttggc 190 374  19S_targ_NP_Lsh/Lw2/Ca/LbFSL_19 tgttacccccatccaacagggctgcccc 191 375  20S_targ_NP_Lsh/Lw2/Ca/LbFSL_20 ctaaagttatagccagaaatgttgatgc 192 376  21S_targ_NP_Lsh/Lw2/Ca/LbFSL_21 ccccagaactgggtgcttgtctttcagc 193 377  22S_targ_NP_Lsh/Lw2/Ca/LbFSL_22 gatacttgactgtgtaaagcaagccaag 194 378  23S_targ_NP_Lsh/Lw2/Ca/LbFSL_23 tcattgagcggagtctgtgactgtttgg 195 379  24S_targ_NP_Lsh/Lw2/Ca/LbFSL_24 tggcattgtgccaaattgattgttcaaa 196 380  25S_targ_NP_Lsh/Lw2/Ca/LbFSL_25 aaactcttaccacaccacttgcaccctg 197 381  26S_targ_NP_Lsh/Lw2/Ca/LbFSL_26 atctgtaggatctgagatctttggtcta 198 382  27S_targ_NP_Lsh/Lw2/Ca/LbFSL_27 catatatacccctgaagcctggggcctt 199 383  28S_targ_NP_Lsh/Lw2/Ca/LbFSL_28 tcagcttctcaaggtcagccgcaagaga 200 384  29S_targ_NP_Lsh/Lw2/Ca/LbFSL_29 ctccccactttcaaaacattcttctttg 201 385  30S_targ_NP_Lsh/Lw2/Ca/LbFSL_30 agaatgtacagtctggttgagacttctg 202 386  31S_targ_NP_Lsh/Lw2/Ca/LbFSL_31 ctctcttttccttcctcatgatcctctg 203 387  32S_targ_NP_Lsh/Lw2/Ca/LbFSL_32 tccaacccattcagaaggttggttgcat 204 388  33S_targ_NP_Lsh/Lw2/Ca/LbFSL_33 atctgatgtgaagctctgcaattctctt 205 389  34S_targ_NP_Lsh/Lw2/Ca/LbFSL_34 agctcttaacttccttagacaaggacat 206 390  35S_targ_NP_Lsh/Lw2/Ca/LbFSL_35 agacaaatgcgcaatcaaatgcctagga 207 391  START OF Znc   1 L_targ_Znc_Lsh/Lw2/Ca/LbFSL_1cgcaccgaggatcctaggctttttgatg 208 392   2 L_targ_Znc_Lsh/Lw2/Ca/LbFSL_2ttgagggactcaagttgctgtcacgctg 209 393   3 L_targ_Znc_Lsh/Lw2/Ca/LbFSL_3gttacaagctgatagacaattctctcat 210 394   4 L_targ_Znc_Lsh/Lw2/Ca/LbFSL_4tagtagatggtcgttgtgattatgataa 211 395 START of Lnc   1L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_1 cgcaccgaggatcctaggctttttgatg 212 396   2L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_2 ttgagggactcaagttgctgtcacgctg 213 397   3L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_3 gttacaagctgatagacaattctctcat 214 398   4L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_4 tagtagatggtcgttgtgattatgataa 215 399   5L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_5 tgaagactactatcgacaagcgctccgg 216 400   6L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_6 ctcttttcagcaggtttagaagagattt 217 401   7L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_7 cagtggaatcctgtcctttgatgagatt 218 402   8L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_8 tgttaaatctggatgttttgtgtctttc 219 403   9L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_9 gacttttgaggaatagaaaaaagtcaaa 220 404  10L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_10 accttgagcaagaatgccacataccatt 221 405  11L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_11 tatcatctgtttgtctggccttaacaaa 222 406  12L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_12 acctattataccagaagactggagaatc 223 407  13L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_13 tttcatggattagatcatgtcctgattt 224 408  14L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_14 tgtcagatttctcatctacatcattaat 225 409  15L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_15 ttatgttgaatgtgtcatacctgtgtca 226 410  16L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_16 aagaatgtgtgttctatgagcaaatgaa 227 411  17L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_17 taaacagcctagatcccatgactaactc 228 412  18L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_18 ttacggagagtttcgtaagaaaacaaaa 229 413  19L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_19 atggagaagaagagacaagcttcttcaa 230 414  20L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_20 taacagggaagatggctcacttaagaaa 231 415  21L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_21 atttcacctcagtcattaaagatcagtg 232 416  22L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_22 atatgcaaaggcaaggcttgaatttcaa 233 417  23L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_23 tctactcagaagaatcaccaacatcata 234 418  24L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_24 aagattattttgagtctttttcaagttt 235 419  25L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_25 tgtgcccatttttgttcttaatgtttct 236 420  26L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_26 tgaaatcatatgtcaagtcgaagctaaa 237 421  27L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_27 cttctgacttctatggtttgcttagcga 238 422  28L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_28 aggaagtccttgtcctgttggaattcca 239 423  29L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_29 ttgtatctgcagcgctacacaatgtcaa 240 424  30L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_30 atgccaatttccctttggatccatttct 241 425  31L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_31 tgcatcataagctcaaaaatggtgaatt 242 426  32L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_32 tgaatgaaatgttcccattaaggatggt 243 427  33L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_33 tgtcagaagcaatcaacaagtcagcttt 244 428  34L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_34 atcatgtgacaagagtttgcaatcggga 245 429  35L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_35 tgggtgtttgggtcctagcagaaccgac 246 430  36L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_36 aatctgtgaggcggttatatcccaagat 247 431  37L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_37 tcatttctcatgttgttaagtggaaaag 248 432  38L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_38 tgtattttgaatcatttgttcgagaatt 249 433  39L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_39 aaaatgtggagaggcctatgtttaggaa 250 434  40L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_40 gggactacatgctcaactacacaaaagg 251 435  41L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_41 ttttatcatgctctcacctctttaaggg 252 436  42L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_42 ccttgatacttggtgattctcttgagct 253 437  43L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_43 accttgtccaaaatatcattgtgaagct 254 438  44L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_44 taagtgtaatcctccaggaactatgtat 255 439  45L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_45 ttagactcaaggggaggtcctgcgacga 256 440  46L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_46 gtgggtgtgtgtgtgtgtgtgtgtgcgt 257 441  47L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_47 tgttgatatcttcaatctggttggtaat 258 442  48L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_48 taaagggccaagataggtggtatctggt 259 443  49L_targ_Lnc_Lsh/Lw2/Ca/LbFSL_49 caactaaacgcctaggatccccggtgcg 260 444START OF GPCnc   1 S_targ_GPCnc_Lsh/Lw2/Ca/LbFSL_1cgcaccggggatcctaggctttttggat 261 445   2 S_targ_GPCnc_Lsh/Lw2/Ca/LbFSL_2tgcttatcgtgatcacgggtatcaaggc 262 446   3 S_targ_GPCnc_Lsh/Lw2/Ca/LbFSL_3agtcagtggagtttgatatgtcacatct 263 447   4 S_targ_GPCnc_Lsh/Lw2/Ca/LbFSL_4tgacctctgccttcaataaaaagacctt 264 448   5 S_targ_GPCnc_Lsh/Lw2/Ca/LbFSL_5caaatgcacaaagtgctcagagccagtg 265 449   6 S_targ_GPCnc_Lsh/Lw2/Ca/LbFSL_6accaatacctgattatacaaaatagaac 266 450   7 S_targ_GPCnc_Lsh/Lw2/Ca/LbFSL_7cttcaggggtggagaatccaggtggtta 267 451   8 S_targ_GPCnc_Lsh/Lw2/Ca/LbFSL_8acaaggctgctttgagtaagttcaaaga 268 452   9 S_targ_GPCnc_Lsh/Lw2/Ca/LbFSL_9tttggtacctagaacatgcaaagaccgg 269 453  10S_targ_GPCnc_Lsh/Lw2/Ca/LbFSL_10 acataaagaggcaggggagtacccccct 270 454 11 S_targ_GPCnc_Lsh/Lw2/Ca/LbFSL_11 taaccaacaaaggaatttgtagttgtgg 271455 START OF NPnc   1 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_1cgcacagtggatcctaggcatttgattg 272 456   2 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_2tgtcattaaggatgcaaccaaccttctg 273 457   3 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_3aaagtcaacatcaaagaagaatgttttg 274 458   4 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_4aacacagcaactagaccaaagatctcag 275 459   5 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_5ggcttgtatggccaaacagtcacagact 276 460   6 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_6acagcagtccagcatcaacatttctggc 277 461   7 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_7ggccaagagaaaactcaacatgtttgtt 278 462   8 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_8aacaattgatctcacaagcgagaaacct 279 463   9 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_9gattgacattgagggtagatttaatgat 280 464  10 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_10cctcttcaatgcgcaacccgggttgacc 281 465  11 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_11cagggaagcttcgagggagtatgaagac 282 466  12 S_targ_NPnc_Lsh/Lw2/Ca/LbFSL_12caaagcaaggctcccagatctgaaaact 283 467

To perform the screen, HEK293FT cells were reverse transfected with aplasmid encoding Cas13a with BFP fluorescence and a plasmid encoding asingle guide using lipofectamine 2000. Three control guides were alsoreverse transfected: 1 empty vector, and 2 off-target guides.Twenty-four hours after transfection, cells were infected with aGFP-expressing LCMV at an MOI 1 for 1 hour. Viral replication, asmeasured by GFP fluorescence, was then measured 48 hours post infection(72 hours post reverse transfection). The fraction of guides whichreduce viral replication is provided in FIG. 5 and Table 14 below. Theamount of viral reduction, as measured by fold change in GFPfluorescence, is provided in FIG. 6. FIGS. 7A and 7B providerepresentative images illustrating the reduction of GFP. From FIG. 8 itis clear that targeting guides cluster along the LCMV genome.

TABLE 14 Total Total Targeting guides guides targeting Targeting guides(in non-coding Protein tested guides (in coding region) region) GPC 43 65 1 NP 47 8 7 1 Z 11 0 0 0 L 182 39 34 5 Totals 283 53 46 7

Various modifications and variations of the described methods,pharmaceutical compositions, and kits of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific embodiments, it will be understood that it iscapable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention. This application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure come within known customarypractice within the art to which the invention pertains and may beapplied to the essential features herein before set forth.

1. A Class 2, type VI CRISPR system comprising (a) a Class 2, type VICRISPR effector protein and/or a polynucleic acid encoding said effectorprotein and (b) one or more guide RNAs and/or one or more polynucleicacids encoding said one or more guide RNAs designed to bind to one ormore target molecules of a virus for use in treating, preventing,suppressing, and/or alleviating viral pathogenesis, infection,propagation and/or replication in a subject.
 2. The CRISPR system foruse according to claim 1, wherein said polynucleic acid encoding saideffector protein comprises a regulatory element operably linked to apolynucleic acid encoding said effector protein.
 3. The CRISPR systemfor use according to claim 1, wherein said polynucleic acid encodingsaid one or more guide RNAs comprises a regulatory element operablylinked to a polynucleic acid encoding said one or more guide RNAs. 4.The CRISPR system for use according to claim 1, wherein said polynucleicacid encoding said one or more guide RNAs and/or said effector proteinare comprised in one or more vectors, preferably (eukaryotic) expressionvectors.
 5. The CRISPR system for use according to claim 4, wherein saidvector is a viral vector, optionally an adenoviral vector, an AAVvector, or a retroviral vector.
 6. (canceled)
 7. The CRISPR system foruse according to claim 1 for use in reducing viremia.
 8. The CRISPRsystem for use according to claim 1, wherein said subject is an animalsubject, optionally a mammalian subject, or optionally a human subject.9. (canceled)
 10. The CRISPR system for use according to claim 1,wherein said target molecule comprises RNA and is part of said virus oris transcribed from a DNA molecule of said virus, wherein the CRISPRsystem effector protein is a RNA-targeting effector protein, and whereinsaid effector protein cleaves said target molecule.
 11. (canceled) 12.(canceled)
 13. (canceled)
 14. The CRISPR system for use according toclaim 1, wherein said effector protein comprises one or more mutations,and wherein the one or more mutations affect catalytic activity and/orstability and/or specificity.
 15. (canceled)
 16. The CRISPR system foruse according to claim 1, wherein said effector protein is codonoptimized, wherein said effector protein optionally comprises a NLS or aNES, and wherein said effector protein optionally comprises a fusionprotein.
 17. (canceled)
 18. (canceled)
 19. The CRISPR system for useaccording to claim 2, wherein said regulatory element allowsconstitutive or inducible expression of said effector protein and/orsaid one or more guide RNAs, optionally tissue specific expression. 20.The CRISPR system for use according to claim 10, wherein saidRNA-targeting effector protein comprises one or more HEPN domains thatcomprise a RxxxxH motif sequence, wherein the RxxxxH motif sequencecomprises a R{N/H/K]X1X2X3H sequence, and wherein X1 is R, S, D, E, Q,N, G, or Y, X2 is independently I, S, T, V, or L, and X3 isindependently L, F, N, Y, V, I, S, D, E, or A.
 21. (canceled) 22.(canceled)
 23. (canceled)
 24. The CRISPR system for use according toclaim 10, wherein the CRISPR RNA-targeting effector protein is Cas13a,Cas13b, or Cas 13c.
 25. The CRISPR system for use according to claim 24,wherein the CRISPR RNA-targeting effector is from an organism of a genusselected from the group consisting of: Leptotrichia, Listeria,Corynebacter, Sutterella, Legionella, Treponema, Filifactor,Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides,Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma, Campylobacter, and Lachnospira, optionallywherein the Cas13 effector protein is from an organism selected from thegroup consisting of: Leptotrichia shahii; Leptotrichia wadei (Lw2);Listeria seeligeri; Lachnospiraceae bacterium MA2020; Lachnospiraceaebacterium NK4A179; Clostridium aminophilum DSM 10710; Carnobacteriumgallinarum DSM 4847; Carnobacterium gallinarum DSM 4847 (second CRISPRLoci); Paludibacter propionicigenes WB4; Listeria weihenstephanensis FSLR9-0317; Listeriaceae bacterium FSL M6-0635; Leptotrichia wadei F0279;Rhodobacter capsulatus SB 1003; Rhodobacter capsulatus R121; Rhodobactercapsulatus DE442; Leptotrichia buccalis C-1013-b; Herbinixhemicellulosilytica; Eubacterium rectale; Eubacteriaceae bacteriumCHKCI004; Blautia sp. Marseille-P2398; and Leptotrichia sp. oral taxon879 str. F0557, Lachnospiraceae bacterium NK4A144; Chloroflexusaggregans; Demequina aurantiaca; Thalassospira sp. TSL5-1;Pseudobutyrivibrio sp. OR37; Butyrivibrio sp. YAB3001; Blautia sp.Marseille-P2398; Leptotrichia sp. Marseille-P3007; Bacteroides ihuae;Porphyromonadaceae bacterium KH3CP3RA; Listeria riparia; andInsolitispirillum peregrinum, preferably wherein the Cas13 effectorprotein is a L. wadei F0279 Cas13a effector protein.
 26. (canceled) 27.(canceled)
 28. The CRISPR system for use according claim 1, wherein theone or more guide RNAs designed to bind to the target molecules comprisea (synthetic) mismatch, and wherein said mismatch is up- or downstreamof a SNP or other single nucleotide variation in said target molecule.29. (canceled)
 30. The CRISPR system for use according to claim 1,wherein the guide RNAs comprise a pan-viral guide RNA set that targetseach virus and/or viral strain in a set of viruses.
 31. The CRISPRsystem for use according to claim 1, wherein the virus is a DNA virus,optionally a single stranded or double stranded DNA virus, optionally apositive sense DNA virus or a negative sense DNA virus or an ambisenseDNA virus, optionally wherein the guide RNA binds to the coding strandor the non-coding strand, preferably the coding strand.
 32. (canceled)33. (canceled)
 34. The CRISPR system for use according to claim 31,wherein the virus is a is a Myoviridae, Podoviridae, Siphoviridae,Alloherpesviridae, Herpesviridae (including human herpes virus, andVaricella Zoster virus), Malocoherpesviridae, Lipothrixviridae,Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae(including African swine fever virus), Baculoviridae, Cicaudaviridae,Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae,Guttaviridae, Hytrosaviridae, Iridoviridae, Maseilleviridae,Mimiviridae, Nudiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae,Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae(including Simian virus 40, JC virus, BK virus), Poxviridae (includingCowpox and smallpox), Sphaerolipoviridae, Tectiviridae, Turriviridae,Dinodnavirus, Salterprovirus, Rhizidovirus, or combination thereof. 35.The CRISPR system for use according to claim 1, wherein the virus is asingle-stranded or double-stranded RNA virus, optionally a positivesense RNA virus or a negative sense RNA virus or an ambisense RNA virus,optionally wherein the guide RNA binds to the coding strand or thenon-coding strand, preferably the coding strand.
 36. (canceled) 37.(canceled)
 38. The CRISPR system for use according to claim 35, whereinthe virus is a Retroviridae virus, Lentiviridae virus, Coronaviridaevirus, a Picornaviridae virus, a Caliciviridae virus, a Flaviviridaevirus, a Togaviridae virus, a Bornaviridae, a Filoviridae, aParamyxoviridae, a Pneumoviridae, a Rhabdoviridae, an Arenaviridae, aBunyaviridae, an Orthomyxoviridae, or a Deltavirus, optionally whereinthe virus is Lymphocytic choriomeningitis virus, Coronavirus, HIV, SARS,Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, Yellow fever virus,West Nile virus, Hepatitis C virus, Dengue fever virus, Zika virus,Rubella virus, Ross River virus, Sindbis virus, Chikungunva virus, Bornadisease virus, Ebola virus, Marburg virus, Measles virus, Mumps virus,Nipah virus, Hendra virus, Newcastle disease virus, Human respiratorysyncytial virus, Rabies virus, Lassa virus, Hantavirus, Crimean-Congohemorrhagic fever virus, Influenza, or Hepatitis D virus.
 39. (canceled)40. A pharmaceutical composition comprising the CRISPR system as definedin claim 1, for use in treating, preventing, suppressing, and/oralleviating viral pathogenesis, infection, propagation and/orreplication in a subject, or for immunizing a subject, or for reducingviremia or viral load in a subject, optionally further comprising one ormore pharmaceutically acceptable excipients.
 41. (canceled)
 42. A methodfor treating, preventing, suppressing, and/or alleviating viralpathogenesis, infection, propagation and/or replication in a subject,comprising administering to a subject in need thereof a Class 2, type VICRISPR system comprising (a) a Class 2, type VI CRISPR effector proteinand/or a polynucleic acid encoding said effector protein and (b) one ormore guide RNAs and/or one or more polynucleic acids encoding said oneor more guide RNAs designed to bind to one or more target molecules of avirus.
 43. The method according to claim 42, wherein said polynucleicacid encoding said effector protein comprises a regulatory elementoperably linked to a polynucleic acid encoding said effector protein.44. The method according to claim 42, wherein said polynucleic acidencoding said one or more guide RNAs comprises a regulatory elementoperably linked to a polynucleic acid encoding said one or more guideRNAs.
 45. The method according to claim 42, wherein said polynucleicacid encoding said one or more guide RNAs and/or said effector proteinare comprised in one or more vectors, preferably (eukaryotic) expressionvectors.
 46. The method according to claim 45, wherein said vector is aviral vector, preferably an adenoviral vector, an AAV vector, or aretroviral vector.
 47. (canceled)
 48. The method according to claim 42for use in reducing viremia.
 49. The method according to claim 42,wherein said subject is an animal subject, preferably a mammaliansubject or a human subject.
 50. (canceled)
 51. The method according toclaim 42, wherein the CRISPR system effector protein is a RNA-targetingeffector protein, and wherein said target molecule comprises RNA. 52.(canceled)
 53. The method according to claim 42, wherein said targetmolecule is part of said virus or is transcribed from a DNA molecule ofsaid virus.
 54. The method according to claim 42, wherein said effectorprotein cleaves said target molecule.
 55. The method according to claim42, wherein said effector protein comprises one or more mutations, andwherein the one or more mutations affect catalytic activity and/orstability and/or specificity.
 56. (canceled)
 57. The method according toclaim 42, wherein said effector protein is codon optimized, wherein saideffector protein optionally comprises a NLS or a NES, and wherein saideffector protein optionally comprises a fusion protein.
 58. (canceled)59. (canceled)
 60. The method according to claim 43, wherein saidregulatory element allows constitutive or inducible expression of saideffector protein and/or said one or more guide RNAs, optionally tissuespecific expression.
 61. The method according to claim 51, wherein saidRNA-targeting effector protein comprises one or more HEPN domains thatcomprises a RxxxxH motif sequence, wherein the RxxxxH motif sequencecomprises a R{N/H/K]X1X2X3H sequence, and wherein X1 is R, S, D, E, Q,N, G, or Y, X2 is independently I, S, T, V, or L, and X3 isindependently L, F, N, Y, V, I, S, D, E, or A.
 62. (canceled) 63.(canceled)
 64. (canceled)
 65. The method according to claim 51, whereinthe CRISPR RNA-targeting effector protein is a Cas13 effector protein.66. The method according to claim 65, wherein the Cas13 effector proteinis from an organism of a genus selected from the group consisting of:Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema,Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma,Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma, Campylobacter, and Lachnospira, optionallywherein the Cas13 effector protein is from an organism selected from thegroup consisting of: Leptotrichia shahii; Leptotrichia wadei (Lw2);Listeria seeligeri; Lachnospiraceae bacterium MA2020; Lachnospiraceaebacterium NK4A179; Clostridium aminophilum DSM 10710; Carnobacteriumgallinarum DSM 4847; Carnobacterium gallinarum DSM 4847 (second CRISPRLoci); Paludibacter propionicigenes WB4; Listeria weihenstephanensis FSLR9-0317 Listeriaceae bacterium FSL M6-0635; Leptotrichia wadei F0279Rhodobacter capsulatus SB 1003; Rhodobacter capsulatus R121; Rhodobactercapsulatus DE442; Leptotrichia buccalis C-1013-b: Herbinixhemicellulosilytica; Eubacterium rectale; Eubacteriaceae bacteriumCHKCI004; Blautia sp. Marseille-P2398; and Leptotrichia sp. oral taxon879 str. F0557, Lachnospiraceae bacterium NK4A144; Chloroflexusaggregans: Demequina aurantiaca; Thalassospira sp. TSL5-1;Pseudobutyrivibrio sp. OR37; Butyrivibrio sp. YAB3001; Blautia sp.Marseille-P2398; Leptotrichia sp. Marseille-P3007; Bacteroides ihuae;Porphyromonadaceae bacterium KH3CP3RA; Listeria riparia; andInsolitispirillum peregrinum, preferably wherein the Cas13 effectorprotein is a L. wadei F0279 Cas13a effector protein.
 67. (canceled) 68.(canceled)
 69. The method according to claim 42, wherein the one or moreguide RNAs designed to bind to the target molecules comprise a(synthetic) mismatch, and wherein said mismatch is up- or downstream ofa SNP or other single nucleotide variation in said target molecule. 70.(canceled)
 71. The CRISPR system according to claim 42, wherein theguide RNAs comprise a pan-viral guide RNA set that targets each virusand/or viral strain in a set of viruses.
 72. The method according toclaim 42, wherein the virus is a DNA virus, optionally a single-strandedor double-stranded DNA virus, optionally a positive sense DNA virus or anegative sense DNA virus or an ambisense DNA virus, optionally whereinthe guide RNA binds to the coding strand or the non-coding strand,preferably the coding strand.
 73. (canceled)
 74. (canceled)
 75. Themethod according to claim 72, wherein the virus is a is a Myoviridae,Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae (includinghuman herpes virus, and Varicella Zoster virus), Malocoherpesviridae,Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae,Ascoviridae, Asfarviridae (including African swine fever virus),Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae,Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae,Iridoviridae, Maseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae,Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae,Polydnaviruses, Polyomaviridae (including Simian virus 40, JC virus, BKvirus), Poxviridae (including Cowpox and smallpox), Sphaerolipoviridae,Tectiviridae, Turriviridae, Dinodnavirus, Salterprovirus, Rhizidovirus,or combination thereof.
 76. The method according to claim 42, whereinthe virus is an RNA virus, optionally a single-stranded ordouble-stranded RNA virus, optionally wherein the virus is a positivesense RNA virus or a negative sense RNA virus or an ambisense RNA virus,optionally wherein the guide RNA binds to the coding strand or thenon-coding strand, preferably the coding strand.
 77. (canceled) 78.(canceled)
 79. The method according to claim 76, wherein the virus is aRetroviridae virus, Lentiviridae virus, Coronaviridae virus, aPicornaviridae virus, a Caliciviridae virus, a Flaviviridae virus, aTogaviridae virus, a Bornaviridae, a Filoviridae, a Paramyxoviridae, aPneumoviridae, a Rhabdoviridae, an Arenaviridae, a Bunyaviridae, anOrthomyxoviridae, or a Deltavirus, optionally wherein the virus isLymphocytic choriomeningitis virus, Coronavirus, HIV, SARS, Poliovirus,Rhinovirus, Hepatitis A, Norwalk virus, Yellow fever virus, West Nilevirus, Hepatitis C virus, Dengue fever virus, Zika virus, Rubella virus,Ross River virus, Sindbis virus, Chikungunva virus, Borna disease virus,Ebola virus, Marburg virus, Measles virus, Mumps virus, Nipah virus,Hendra virus, Newcastle disease virus, Human respiratory syncytialvirus, Rabies virus, Lassa virus, Hantavirus, Crimean-Congo hemorrhagicfever virus, Influenza, or Hepatitis D virus.
 80. (canceled)
 81. Use ofthe CRISPR system as defined in claim 1 for the manufacture of amedicament for treating, preventing, suppressing, and/or alleviatingviral pathogenesis, infection, propagation and/or replication in asubject.
 82. The CRISPR system for use according to claim 1, whereinsaid one or more guide RNAs is two or more, three or more, four or more,or five or more gRNAs.
 83. (canceled)
 84. (canceled)
 85. (canceled) 86.The method or CRISPR system for use according to claim 82, wherein saidguide RNAs bind to different target sequences.
 87. The method or CRISPRsystem for use according to claim 82, wherein said one or more guideRNAs are selected based on prior diagnosis or detection of the virus,and wherein said diagnosis or detection comprises identifying aparticular viral mutation or nucleotide variation.
 88. (canceled) 89.The method or CRISPR system for use according to claim 87, wherein saiddiagnosis or detection comprises identifying a particular viral strainor particular viral drug resistance.
 90. (canceled)
 91. The method orCRISPR system for use according to claim 87, wherein said diagnosis ordetection comprises a companion or complementary diagnostic method. 92.The method according to claim 42, wherein said one or more guide RNAs istwo or more, three or more, four or more, or five or more gRNAs.
 93. Themethod according to claim 92, wherein said guide RNAs bind to differenttarget sequences.
 94. The method according to claim 92, wherein said oneor more guide RNAs are selected based on prior diagnosis or detection ofthe virus, and wherein said diagnosis or detection comprises identifyinga particular viral mutation or nucleotide variation.